JP6537514B2 - Flavor stable beverage - Google Patents

Description

  The present invention relates to a method for producing a flavor stable beverage. The method of the invention comprises the step of reducing the content of amino acids in the beverage, resulting in a beverage with a much reduced generation of ripening flavor. The invention also provides a useful method to reduce the content of amino acids in the beverage without adversely affecting the flavor of the beverage.

The flavor profile of beer undergoes changes during storage. Strecker aldehyde has been considered to be an important component of the ripening flavor in beer. Strecker aldehyde is proposed to be formed from amino acids, at least in part, by transamination, which occurs between amino acids and α-dicarbonyls. In particular, it is proposed that the amino acids listed in Table 1 may be involved in the formation of streckeraldehyde.

  The flavor threshold for these aldehydes was determined by Meilgaard in 1975 in Flavor chemistry in beer: Part II: Flavor and flavor threshold of 239 aroma volatiles, Tech. Q. -Master Brew. Assoc. Am. , 12: 151-168 and is shown in Table 1. The addition of amino acids to beer appears to result in an increase in the level of streckeraldehyde. Thus, Vesely et al., 2003 (The 29th Annual European Brewing Agenda Conference-(2003), 94 / 1-94 / 11) found an increase in the level of various strecker aldehydes after adding amino acids to beer The

  However, the involvement of amino acids in the formation of ripening flavors in beer under normal conditions has been suspected. Thus, in one study, 85% of the Strecker aldehyde present in aged beer was derived from Strecker degradation during wort production, while only 15% was derived from Strecker degradation in bottled beer (Suda et al., 31st Annual Meeting of the European Brewery Convention (2007), suda1 / 1-suda1 / 7) were found. Thus, amino acid levels in fresh beer appear to have little effect on the formation of streckeraldehyde during beer ripening.

  Aldehydes formed during beer production are also found to be linked to other compounds, which can be released over time during storage (Baert et al., 2012, J. Agric. Food Chem., 60: 1 1449-11472). Thus, attempts to reduce strecker aldehyde in beer have been aimed at reducing the formation and / or content of aldehyde during the manufacture of beer. Baert et al. 2012 (see above) thus describes a number of practical means for reducing aldehyde aging in beer, including, for example, the use of yeast strains with high aldehyde reduction activity.

  Thus, there is a need for a method for preparing beer and other cereal-based beverages, wherein the formation of ripening flavors is significantly reduced in said beer during storage. The inventors have found that by reducing the level of amino acids in the wort, so that the formation of the ripening flavor is significantly reduced during storage in the resulting beverage.

  In contrast, previous strategies for reducing Strecker aldehyde have usually been directed to reducing the level of aldehyde itself during beer production. Thus, Baert et al. 2012 (see above) describes a number of methods to reduce aldehyde aging in beer, but one of these is directed to reducing the amount of amino acids in wort or in fermentation. There is nothing to be done.

  However, the invention provides a method for preparing a beverage from cereal extracts containing high or medium levels of amino acids, wherein the method comprises the step of reducing the level of Strecker amino acids, during storage A beverage is obtained which does not develop a ripening flavor or only to a much lower extent.

  The invention also provides a number of novel methods for reducing the level of amino acids in cereal extracts.

Thus, one aspect of the present invention is to provide a method for producing a flavor-stable cereal-based beverage, said method comprising the following steps:
i) providing a cereal extract comprising at least 25 mg / L methionine;
ii) treating the cereal extract to reduce the content of one or more amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine, thereby obtaining a treated cereal extract;
iii) processing the processed cereal extract into a beverage,
Here, the beverage has at most 100 mg / L of amino acids, a total content of methionine, phenylalanine, valine, leucine and isoleucine and / or a content of methionine of less than 5 mg / L.

It is also an aspect of the invention to provide a method for reducing the content of at least one amino acid in cereal extract, said method comprising the following steps:
a) increasing the level of acid and / or base in the cereal extract; and b) at least a portion of the acid anion of the increased acid and / or the basic cation of the increased base Removing through the reinforced dialysis membrane stack.

It is also an aspect of the invention to provide a method for producing a flavor stable cereal based beverage, said method comprising the following steps:
i) providing cereal extract;
ii) treating said cereal extract so as to reduce the content of at least one amino acid by performing the method comprising steps a) and b) described above, whereby the treated cereal extract is obtained Obtaining process;
iii) processing the processed cereal extract into a beverage.

  Furthermore, it is an aspect of the invention to provide a method for reducing the content of methionine in cereal extract, said method comprising the step of incubating said cereal extract with an oxidizing agent.

It is also an aspect of the invention to provide a method for reducing the content of methionine in cereal extracts, said method being capable of catalyzing the cereal extract to form H ₂ O ₂ Incubate with the enzyme or mixture of enzymes.

It is also an aspect of the invention to provide a method for producing a cereal based beverage, said method comprising the following steps:
i) providing cereal extracts comprising methionine;
ii) treating the cereal extract to reduce the content of methionine as described above, thereby obtaining a treated cereal extract;
iii) processing the processed cereal extract into a beverage.

The contents of methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe) during REED coenzyme conversion of glucose to gluconic acid are shown. The test was performed at 40 ° C. and pH 4.2 and ended after about 11 hours. Figure 10 shows the percentage recovery of amino acids on wort after the REED coenzyme process converting glucose to gluconic acid. Both tests were performed at 40 ° C. and ended after about 11 hours. In Test A, the pH set point was 4.2. In Test B, the pH set point was 5.5. Lc. Reduction of the content of methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe) in wort during REED-assisted fermentation of glucose to lactic acid using lactis Show. The fermentation was performed at 37 ° C. and pH 5.5. Lc. Reduction of the content of methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe) in wort during REED-assisted fermentation of glucose to lactic acid using lactis Show. The fermentation was carried out at 30 ° C. and pH 5.5 on a 150 L scale. Figure 7 shows the reduction of the content of methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe) in wort during titration and pH control of lactic acid using REED. 5 L scale at 40 ° C. and pH 5.5. The contents of amino acids methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe) in glucose wort are shown during incubation with hydrogen peroxide at 40 ° C. Upper panel: control-wort incubated without H ₂ O ₂ . Middle panel: 50 ppm of H ₂ O ₂ at the start. Bottom panel: 250 ppm H ₂ O ₂ at the start. The scores for the individual ripening flavors on a scale of 0 to 5 for REED-beverage DS are shown. A: Beverage DS, stored at 20 ° C. for 2 months B: Beverage DS, stored at 20 ° C. for 6 months. 3 shows an exemplary REED installation. L. Figure 7 shows the reduction in the content of methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe) in wort during REED assisted fermentation with L. lactis. The fermentation was performed at 37 ° C. and pH 6.0. The Strecker aldehyde forming amino acid is removed after 13 hours of fermentation.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing cereal-based beverages which are less likely to develop ripening flavors during storage. Interestingly, the present invention discloses that the beverage subjected to the step of reducing the amino acid content is much less likely to develop a ripening flavor. The properties of the beverage prepared by the method of the invention are described in more detail hereinafter in the section "Properties of the Beverage".

In general, the method according to the invention comprises the following steps:
i) providing cereal extract ii) treating the cereal extract so as to reduce the content of one or more amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine Step iii) of processing the processed cereal extract into a beverage.

  Step i) consists of providing a cereal extract, which may be any of the cereal extracts described hereinafter in the section "cereals extract". Cereal extracts contain relatively high levels of amino acids, however, these can be reduced by simple methods such as dilution with water or an aqueous solution containing, for example, sugar. However, dilution has an adverse effect on the taste of the resulting beverage and is therefore undesirable. The methods of the present invention are most useful for preparing medium to high levels of cereal extract beverages with amino acids. Thus, preferably, the cereal extract comprises at least 25 mg / L methionine. An example of such cereal extract is a common malt based wort composition.

Step ii) comprises treating the cereal extract to reduce the content of one or more amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine. Said treatment may be carried out by reducing the level of amino acids with the aid of dialysis described hereinafter in the section "Reducing the content of amino acids by dialysis". In particular, the invention increases the level of at least one acid, while at the same time reducing the level of acidic anions, at the same time, through an anion exchange reverse electrical enhancement dialysis (AX-REED) membrane stack, It is disclosed that the level of amino acids can be reduced. By increasing the level of at least one base, while at the same time at the same time reducing the level of basic cations by passing through the cation exchange reverse electro-reinforcement dialysis (CX-REED) membrane stack, the level of amino acids is increased It can be reduced. The level of amino acids may also be reduced with the aid of the oxidizing agents described hereinafter in the section "Reducing the content of amino acids by oxidizing agents". Interestingly, the present invention discloses that the level of some Strecker amino acids, in particular methionine, can be significantly reduced by incubation with an oxidizing agent such as H ₂ O ₂ .

  Step iii) comprises processing the processed cereal extract into a beverage. In some embodiments of the invention, the processed cereal extract may then already be a beverage, in which case step iii) may be omitted.

  However, in most cases, an additional step is required before the processed cereal extract becomes a beverage. In one embodiment of the invention, the processed cereal extract may be subjected to conventional fermentation, for example conventional alcoholic fermentation. In such cases, the beverage may be beer. In particular, if step ii) comprises or consists of reducing the content of amino acids by dialysis or reducing the content of amino acids by means of an oxidizing agent.

  Frequently, one or more additional compounds may be added to the processed cereal extract. The additional compound may be, for example, a flavor compound, a preservative or a functional ingredient component. In particular, the additional compound may be any of the additional compounds described herein below in the section "Additional Compounds".

  The processed cereal extract may also be mixed with one or more other liquids before obtaining the final beverage. In one embodiment of the invention, the treated cereal extract is mixed with another cereal extract, for example a wort such as a wort prepared from ground malt, followed by fermentation of said mixture.

  Step iii) may also include the step of carbonizing to obtain a sparkling beverage.

  Step iii) can be carried out in any of the ways described hereinafter in the section "Processing into beverages".

  Interestingly, the beverage prepared by the method of the present invention is less likely to develop a ripening flavor. Thus, the beverage prepared by the method according to the invention can be stored for a long time. As such, the method for preparing the beverage according to the invention may also include step iv) consisting of storing the beverage. Step iv) stores the beverage for at least one week, such as at least 2 weeks, such as at least 1 month, such as at least 2 months, such as at least 3 months, such as at least 3 months, such as at least 6 months, such as at least 12 months Or may be configured from this. In particular step iv) takes at least 1 week, for example at least 2 weeks, such as at least 1 month, such as at least 2 months, such as at least 2 months, such as at least 3 months, such as at least 6 months, such as at least 12 months. At the temperature, it may involve storing the beverage, or be composed of this. The ambient temperature is preferably a temperature in the range of 15-30 ° C., for example a temperature in the range of 20-25 ° C. In another embodiment, step iv) may comprise or consist of storing the beverage at high temperature for at least one month, such as at least two months, such as at least three months, such as at least six months. It can be done. The high temperature is preferably a temperature in the range of 30 to 50 ° C, for example a temperature in the range of 30 to 40 ° C. It should be understood that the storage may be performed for any reason, for example storage may be performed to obtain a particular stored flavor. However, the storage step may also be performed for convenience. Thus, for example, for transportation reasons, the beverage may be stored at the warehouse prior to distribution to a wholesaler or store, and / or it may be stored at the store prior to sale to the end customer, And / or it may be stored by the customer prior to consumption.

Cereal Extract The present invention relates to a method of preparing a beverage comprising step i) of providing cereal extract. In addition, the present invention relates to methods for reducing the content of methionine and / or other Strecker amino acids in cereal extracts. The cereal extract may be any cereal extract, in particular any of the cereal extracts described herein in this section.

  In general, the method is more useful for preparing a beverage of cereal extract containing medium to high levels of amino acids, since the method of the invention comprises the step of reducing the content of at least one amino acid. Sometimes, with cereal extracts that already contain very low levels of amino acids, it may not be advantageous or even required to further reduce the amino acids.

  Preferably, the cereal extract is a pure cereal extract. Pure cereal extracts according to the invention are composed of cereals and / or cereal malt and optionally an aqueous extract of hops. More preferably, the cereal extract is a cereal cereal germ and optionally a pure cereal extract of hops. However, it is also within the scope of the invention that the cereal extract may contain additional auxiliaries. It is also preferred that the cereal extract is a pure cereal extract to which is added one or more compounds selected from the group consisting of salts, acids, bases and buffers.

  Generally, it is preferred that the cereal extract comprises at least 25 mg / L methionine, such as at least 30 mg / L methionine, such as at least 35 mg / L methionine. It is also preferred that the cereal extract comprises at least 90 mg / L valine, such as at least 100 mg / L valine, such as at least 110 mg / L valine. It is also preferred that the cereal extract comprises at least 50 mg / L isoleucine, such as at least 60 mg / L isoleucine, such as at least 70 mg / L isoleucine. It is also preferred that the cereal extract comprises at least 125 mg / L leucine, such as at least 150 mg / L leucine, such as at least 175 mg / L leucine. It is also preferred that the cereal extract comprises at least 90 mg / L phenylalanine, such as at least 110 mg / L phenylalanine, such as at least 130 mg / phenylalanine.

  In general, pure cereal extracts of malt contain the above levels of amino acids unless being diluted. Diluted cereal extracts of malt may be less useful for preparing a beverage, even though the levels of amino acids are reduced, the levels of other compounds are also reduced, eg, Because it contains less sugar and aroma compounds.

  In a preferred embodiment of the invention, the cereal extract is wort, even more preferably, the wort comprises amino acids at the levels mentioned above. In a highly preferred embodiment of the invention, the cereal extract is a malt based wort.

  By the term "wort" is herein meant an aqueous extract of ground cereals. In particular, the wort may be an aqueous extract of ground malt, in which case the wort may be referred to as "wheat-based wort". The wort may also be an aqueous extract of ground malt free cereal grains and / or an aqueous extract of a mixture of ground malt free and malted cereal grains.

  The term "malt" as used herein refers to grain that has been subjected to soaking, germinated and then dried. The drying may be, for example, kiln drying.

  The wort may be prepared from any cereals. The cereals may be selected, for example, from the group consisting of barley, wheat, rye, oats, corn, rice, sorghum, millet, triticale, buckwheat, phonio and quinoa. More preferably, the cereals are selected from the group consisting of barley, wheat, rye, oats, corn and rice, more preferably the cereals are barley.

  Thus, in a preferred embodiment of the invention, the cereal extract is a wort prepared from barley malt. Alternatively, the cereal extract may be wort prepared from malt free barley, or from a mixture of malted and malt free barley.

  Wort is generally prepared by mashing the ground malt with water followed by an optional sparging step. The term "mashing," as used herein, refers to the incubation of ground malt in water. The mashing is preferably carried out at a specific temperature and volume of water. The temperature may be kept constant but is typically changed at specific intervals. The mashing is preferably carried out at a temperature in the range of 50-80 ° C, for example in the range of 60-78 ° C.

  Mashing can occur in the presence of an adjuvant, such as, but not limited to, any source of carbohydrate other than malt, such as barley without barley, barley syrup, or corn, or whole rice or grits, syrups or starches. It is understood that it contains any of the above-mentioned processed products. However, it is preferred that the mashing be carried out without the aid and thus that the wort is also free of the aid.

  The term "sparging" as used herein refers to the process of extracting residual sugars and other compounds with hot water from grains consumed after mashing. Sparsing is typically performed in a filtration tank, mash filter, or another device to allow separation of the extracted water from the consumed grain.

  The wort obtained after mashing is generally referred to as "first wort", while the wort obtained after sparging is generally referred to as "second wort". Unless otherwise stated, the term wort may be primary wort, secondary wort, or a combination of both.

  After sparging, the wort can be heated or boiled, for example in the presence of hops.

  The cereal extract may also be "glucose wort". The term "glucose wort" as used herein refers to wort that has been treated to convert carbohydrates and / or oligosaccharides to glucose either during wort preparation or after wort preparation. . In particular, the glucose wort may be a wort comprising at least 50 g / L, such as at least 80 g / L, such as at least 100 g / L, such as at least 120 g / L glucose.

  Said treatment for converting carbohydrates and / or oligosaccharides to glucose can be carried out using any useful method known to the person skilled in the art, for example, section "Saccharide Glucose to International Patent Application PCT / DK2013 / 052215". Can be carried out by any of the methods described in

Preferably, said treatment of converting carbohydrate and / or oligosaccharide to glucose is carried out with the aid of an enzyme or mixture of enzymes capable of catalyzing the hydrolysis of carbohydrate and / or oligosaccharide to glucose. The enzyme or mixture of enzymes may in particular include:
a) An enzyme capable of catalyzing hydrolysis of the terminal (1 → 4) -linked α-D-glucose residue sequentially from the non-reducing end of the oligosaccharide to obtain release of β-D-glucose, for example , Enzymes classified under EC 3.2.1.3, eg glucan 1,4-α-glucosidase; and / or b) multiple containing three or more (1 → 4) -α-linked D-glucose units Enzymes capable of catalyzing endohydrolysis of (1 → 4) -α-D-glucosidic bonds in saccharides, eg enzymes classified under EC 3.2.1.1, eg α-amylase and / or c) It catalyzes the hydrolysis of (1 → 6) -α-D-glucosidic bonds in pullulan, amylopectin and glycogen, and in amylopectin and α- and β-limited dextrins of glycogen DOO can enzyme, e.g. enzyme classified under EC3.2.1.41, for example, pullulanase.

In one embodiment, the enzyme or mixture of enzymes comprises one or more of the following enzymes:
a) Glucan 1,4-α-glucosidase of SEQ ID NO: 1 of the international patent application PCT / DK2013 / 050215;
b) Glucan 1,4-α-glucosidase of SEQ ID NO: 2 of the International Patent Application PCT / DK2013 / 050215;
c) Glucan 1,4-α-glucosidase of SEQ ID NO: 3 of the international patent application PCT / DK2013 / 050215;
d) the alpha-amylase of SEQ ID NO: 4 of the international patent application PCT / DK2013 / 050215;
e) the alpha-amylase of SEQ ID NO: 5 of the international patent application PCT / DK2013 / 050215;
f) α-Amylase of SEQ ID NO: 6 of the International Patent Application PCT / DK2013 / 050215;
g) Pullulanase of SEQ ID NO: 7 of the International Patent Application PCT / DK2013 / 050215;
h) Pullulanase of SEQ ID NO: 8 of the International Patent Application PCT / DK2013 / 050215;
i) Pullulanase of SEQ ID NO: 9 of the International Patent Application PCT / DK2013 / 050215;
j) Any of the above functional homologues sharing at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity with them.

Treated Cereal Extract The present invention relates to a method of preparing a beverage comprising the step ii) of treating cereal extract to reduce the content of one or more Strecker amino acids. In addition, the invention relates to methods for reducing the content of methionine and / or Strecker amino acids in cereal extracts. Cereal extracts that have been treated to reduce the content of one or more Strecker amino acids are referred to as "processed cereal extracts".

  Depending on the particular method, the processed cereal extract may then contain more or less amino acids. In embodiments of the invention in which the cereal extract is the final beverage or, for example, essentially no amino acids have been removed in step iii), the final beverage is only slightly and / or simply processed from the processed cereal extract It is preferred that the processed cereal extract then contain very low amounts of amino acids, as it can be obtained by the process and they do not significantly alter the amino acid content. Such an embodiment comprises or consists of reducing the content of amino acids by dialysis, wherein the cereal extract is incubated with a microorganism capable of fermenting glucose to an organic acid, A method can be included, wherein at least a portion of the organic acid is removed through an anion exchange reverse electrical enhanced dialysis (AX-REED) membrane stack.

  Thus, in such embodiments, the treated cereal extract is preferably at most 100 mg / L, more preferably at most 50 m / L, still more preferably at most 25 mg / L, even more preferably at most 10 mg / L. Even more preferably, it has a total content of at most 5 mg / L of amino acids methionine, phenylalanine, valine, leucine and isoleucine.

  In particular, the treated cereal extract preferably has a content of methionine of at most 15 mg / L, such as at most 10 mg / L, such as at most 5 mg / L, such as at most 3 mg / L. In some embodiments of the invention, the level of methionine in the processed cereal extract is below the level detected by HPLC. The processed cereal extract may also have a content of valine of at most 15 mg / L, such as at most 10 mg / L, such as at most 5 mg / L. The processed cereal extract may also have a content of isoleucine of at most 15 mg / L, such as at most 10 mg / L, such as at most 5 mg / L. The processed cereal extract may also have a content of leucine of at most 15 mg / L, such as at most 10 mg / L, such as at most 5 mg / L. The processed cereal extract may also have a content of phenylalanine of at most 60 mg / L, such as at most 40 mg / L, such as at most 30 mg / L, such as at most 20 mg / L. In embodiments of the invention where the amino acids are reduced by incubating the cereal extract with the microorganism, at least a portion of which is simultaneously subjected to REED treatment, such levels of amino acids are particularly preferred.

In particular, the invention is characterized by increasing the level of at least one acid or at least one base, while at least one at a time, respectively, at the same time, through an anion exchange reverse electro-reinforcement dialysis (AX-REED) membrane stack. It is disclosed that the level of amino acids can be reduced by reducing the level of acidic anions or reducing the level of said basic cations through a cation exchange reverse electro-enhanced dialysis (CX-REED) membrane stack. The level of amino acids may also be reduced with the aid of an oxidizing agent, which is described herein below in the section "Reducing the content of amino acids by oxidizing agents". Interestingly, the present invention discloses that the level of some Strecker amino acids, in particular methionine, can be significantly reduced by incubation with an oxidizing agent such as H ₂ O ₂ .

  In another embodiment of the invention, the processed cereal extract then preferably has a content of methionine of at most 15 mg / L, such as at most 10 mg / L, such as at most 5 mg / L, such as at most 3 mg / L or even It has a level of methionine below the level detected by HPLC. The processed cereal extract may also have a content of valine of at most 40 mg / L, such as at most 30 mg / L, such as at most 20 mg / L, such as at most 10 mg / L. The processed cereal extract may also have a content of isoleucine of at most 40 mg / L, such as at most 30 mg / L, such as at most 20 mg / L, such as at most 10 mg / L. The processed cereal extract may also have a content of leucine of at most 40 mg / L, such as at most 30 mg / L, such as at most 20 mg / L, such as at most 10 mg / L. The processed cereal extract may also have a content of phenylalanine of at most 40 mg / L, such as at most 30 mg / L, such as at most 20 mg / L. This may be particularly so in embodiments of the invention, where the amino acid increases the level of at least one acid or one base, while at least a portion at the same time in reverse It is reduced by a process comprising reducing the level of acidic anions or basic cations through the electrically reinforced dialysis membrane stack, wherein the acid or base is increased by the addition of the acid or base or with the aid of an enzyme.

  In other embodiments of the invention it may then be preferable for the processed cereal extract to maintain low levels of Strecker amino acids. This is especially so in embodiments of the invention in which step iii) involves microbial fermentation of the treated cereal extract.

  Such embodiments may include methods wherein step ii) comprises or consists of reducing the content of amino acids with an oxidizing agent. Such embodiments also show that step ii) increases the level of the at least one acid or one base, while at least a portion of this simultaneously through the reverse electrical enhanced dialysis membrane stack, the acidic anion or basic cation Comprising or reducing the content of amino acids by a process comprising reducing the level of H, wherein the acid or base is increased with the addition of the acid or base, or with the aid of an enzyme, May include a method.

  In such embodiments, the processed cereal extract may have a total content of at most 300 mg / L, more preferably at most 250 mg / L amino acids, methionine, phenylalanine, valine, leucine and isoleucine. In particular, the treated cereal extract has a content of methionine of at most 15 mg / L, for example at most 10 mg / L, for example at most 5 mg / L, for example at most 3 mg / L, or even methionine levels below the level detected by HPLC. It is preferable to have.

The term amino acid "amino acid" as used herein, amine (-NH ₂₎ and a carboxylic acid acid (-COOH) functional group, a composed organic compound with specific side chains each amino acid. The three letter and one letter codes for the standard amino acids are used according to the IUPAC definition.

  The term "Stretcher amino acid" is used to include the group of amino acids consisting of valine, isoleucine, leucine, methionine and phenylalanine.

  The method of the invention generally comprises the step of reducing the content of amino acids. In particular, it is important that the content of Strecker amino acids be reduced. Therefore, the step of reducing the amino acid content is preferably a step of reducing the content of the Strecker amino acid. In some embodiments, only the content of one Strecker amino acid is reduced. Therefore, the step of reducing the content of amino acids may be reducing the content of one or more amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine, for example, from methionine, phenylalanine, valine, leucine and isoleucine. Reducing the content of at least two amino acids selected from the group consisting of, eg reducing the content of at least three amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine, eg methionine, phenylalanine, Reducing the content of at least four amino acids selected from the group consisting of valine, leucine and isoleucine, eg amino acids, methionine, phenylalanine, valine, leucine and It can include reducing the content of each of isoleucine, or may be constructed from this. In particular, the method of the invention may comprise the step of reducing the content of methionine in the cereal extract. Thus, step ii) of the method may comprise or consist of reducing the level of methionine in the cereal extract.

Reducing the Content of Amino Acids by Dialysis The method of the invention comprises the step of reducing the content of one or more amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine. In one embodiment this may be achieved with the aid of dialysis.

The inventors have found that simple dialysis may not be sufficient to reduce the content of Strecker amino acids, and the level of sugar is not significantly reduced. In fact, even electrodialysis may not be sufficient to specifically reduce the content of Strecker amino acids. However, step ii) may include or be comprised of reducing the content of one or more Strecker amino acids by a method comprising:
a) increasing the level of acid or base in the cereal extract; and b) at least a portion of the acid anion of the increased acid or at least a portion of the basic cation of the increased base. Remove through a reinforced dialysis (REED) membrane stack.

  Removing the acidic anion or the basic cation through the REED membrane stack may be performed in any of the manners described herein below in section "REED".

  In order to maintain and / or control the pH, the method also comprises that the base (step a) comprises increasing the level of acid) or the acid (step a) comprises increasing the level of base) May be included.

  When step a) comprises increasing the level of acid, then step b) preferably comprises passing at least a portion of said increased acidic anions through an anion exchange reverse electrical enhancement dialysis (AX-REED) membrane stack Including removing.

  The removal of the acidic anions through the AX-REED membrane stack may be performed in any of the manners described herein below in section "REED".

  Wherein step a) comprises increasing the level of base, then step b) preferably comprises at least a portion of said increased basic cation, cation exchange reverse electrical enhanced dialysis (CX-REED) membrane stack Including removing through.

  The removal of the basic cation through the CX-REED membrane stack may be performed in any of the manners described herein below in section "REED".

Steps a) and b) may be performed simultaneously, partially simultaneously or sequentially. Preferably, steps a) and b) are performed partially simultaneously, and any of the following methods can be obtained: An acid is added or generated sequentially while simultaneously being removed continuously through the AX-REED membrane stack; Base is added or generated sequentially while simultaneously removing sequentially through the CX-REED membrane stack.

  Frequently, the level of acid or base is increased for some time before removal of the acid anion or basic cation through the REED membrane stack is initiated.

The acid may be any acid, in particular any acid comprising an acidic anion, which is not toxic or otherwise undesirable. Non-limiting examples of useful acids include HCl, phosphoric acid and organic acids. Preferably, the acid is an organic acid. As used herein, the term "organic acid" refers to any carboxylic acid. Preferably, the organic acid according to the present invention is C _1-3 -alkyl or C _1-3 -alkenyl, wherein said C _1-3 -alkyl and C _1-3 -alkenyl are n-COOH groups, substituted with m-OH and q = O, where n is an integer in the range of 1 to 3, m is an integer in the range of 0 to 2 and q is an integer in the range of 0 to 1 .

Preferably, the organic acid may be propyl substituted as follows: 1) 1-3-COOH group, such as 3-COOH group; and 2) 0-1 -OH group, such as 1-OH group or organic acid Are 1) a 1-2-COOH group which may be substituted ethyl, and 2) a 0-OH group.

  Preferably the term "organic acid" is selected herein from the group consisting of lactic acid, citric acid, malic acid, tartaric acid, acetic acid, succinic acid, isocitric acid, alpha-ketoglutaric acid, fumaric acid and oxaloacetic acid. Indicates acid.

  In one highly preferred embodiment of the invention the term "organic acid" as used herein denotes lactic acid.

  The level of acid may be increased using a variety of different methods. Thus, for example, the level may be increased simply by the addition of acid. The acid is preferably added over time, for example, at a rate that does not exceed the ability to extract the corresponding amount of anions through the AX-REED. For example, the rate may be in the range of 2-10 g acid / L / hour. More preferably, a portion of the acid is neutralized by the addition of a base, and then the acid is added at a rate that does not exceed the ability to extract the corresponding amount of anions through AX-REED.

  In another embodiment, the level of acid is increased with the aid of an enzyme capable of catalyzing the formation of acid. This may be performed in any of the ways described herein below in the section "Increasing levels of acid by enzymes". Useful enzymes are also disclosed in that section.

  In another embodiment, the level of acid is increased with the aid of a microorganism capable of fermenting the sugar to an organic acid. This may be performed in any of the ways described herein below in the section "Increasing levels of acid by microorganisms".

  The level of base may also be increased using a variety of different methods. Thus, for example, the level may be increased simply by the addition of a base. The base is preferably added over time, for example, at a rate that does not exceed the ability to extract the corresponding amount of cations through the CX-REED. More preferably, a portion of the base is neutralized by the addition of acid, and then the base is added at a rate that does not exceed the ability to extract the corresponding amount of anions through AX-REED.

Increasing the level of acid by enzymes In one embodiment, the level of acid is increased with the help of an enzyme, preferably an enzyme or mixture of enzymes capable of catalyzing the formation of an organic acid. In particular, the enzyme or mixture of enzymes may be capable of catalyzing the oxidation of sugars to organic acids. Preferably, the enzyme or mixture of enzymes may be capable of catalyzing the oxidation of glucose to an organic acid.

  The enzyme or mixture of enzymes capable of catalyzing the conversion of glucose to form an organic acid can comprise any enzyme capable of catalyzing the conversion of glucose to form an organic acid. In one preferred embodiment of the invention, the enzyme or mixture of enzymes capable of catalyzing the conversion of glucose to form an organic acid comprises or even consists of glucose oxidase.

Glucose oxidase for use with the present invention is an enzyme generally classified under EC 1.1.3.4. Thus, glucose oxidase for use with the present invention is an oxidoreductase capable of catalyzing the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. Thus, in particular, the glucose oxidase used with the present invention is an enzyme capable of catalyzing the following reaction:
β-D-glucose + O ₂ → D-glucono-1,5-lactone + H ₂ O ₂
D-glucono-1,5-lactone is hydrolyzed in water to gluconic acid. Thus, in aqueous environments, gluconic acid is formed by glucose oxidase-catalyzed conversion of glucose. In the method of the invention, an enzyme or mixture of enzymes capable of catalyzing the conversion of glucose to form an organic acid is generally used in an aqueous environment, thus catalyzing the conversion of glucose to form an organic acid The enzyme or mixture of enzymes that can be or can be composed of glucose oxidase.

  Glucose oxidase may be any useful glucose oxidase. For example, the glucose oxidase may be glucose oxidase of Aspergillus niger or Penicillium agama xii. In one embodiment, glucose oxidase shares at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity with glucose oxidase of SEQ ID NO: 1 It is a functional homologue. Glucose oxidase also shares at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity with aa23-605 of SEQ ID NO: 1 or SEQ ID NO: It may comprise or even consist of functional homologues of 1 aa 23-605. Glucose oxidase is also a glucose oxidase of SEQ ID NO: 2 or a functional homologue thereof which shares at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity with this. It may be

  Glucose oxidase is one of commercially available glucose oxidases, such as Amano Pharmaceutical Co. Ltd. And Nagoya, Japan may be Hydelase available from Japan. The advantage of hydelase is that it also includes catalase activity. Thus, hydelase includes both glucose oxidase and catalase activity.

  Furthermore, the glucose oxidases described in GB 1,373,562, US 4,675,191 and US 20120114791 can be used with the present invention.

As mentioned above, reactions catalyzed by glucose oxidase can also lead to the formation of H ₂ O ₂ .

  The enzyme or mixture of enzymes capable of catalyzing the oxidation of sugars to organic acids may be any sugar oxidase. For example, it may be maltose oxidase, which can catalyze the oxidation of maltose to form maltobionic acid. The sugar oxidase may also be lactose oxidase, which can catalyze the conversion of lactose to lactobionic acid.

As disclosed by the present invention, H ₂ O ₂ may be useful to reduce the levels of Strecker amino acids, particularly methionine. Thus, it is preferred that the enzyme or mixture of enzymes capable of catalyzing the formation of acid is also capable of catalyzing the formation of H ₂ O ₂ . As mentioned above, one example of an enzyme capable of catalyzing the oxidation of sugars forming organic acids and catalyzing the formation of H ₂ O ₂ is glucose oxidase.

  It is pointed out here that aerating the cereal extract with oxygen is advantageous during the conversion of glucose to gluconic acid. Aeration has not been found to be detrimental to the taste of the beverage.

  The temperature during the enzymatic conversion of glucose can be chosen such that the enzyme used is active at the selected temperature. The temperature may be, for example, 10 ° C to 70 ° C, such as 20 ° C to 50 ° C.

  Acids, such as gluconic acid formed by enzyme catalysis, can be removed continuously through the AX-REED membrane stack. Thus, the reaction is not inhibited by the accumulation of gluconic acid. This may be performed in any of the ways described herein below in section "REED".

  In a preferred embodiment of the method according to the invention, oxygen is supplied continuously to the cereal extract which is incubated with the enzyme or mixture of enzymes comprising glucose oxidase. The supply of oxygen has a significant beneficial effect on the reaction rate of the enzyme reaction. Thus, the continuous introduction of oxygen ensures a high reaction rate. Oxygen may be supplied by any suitable means, for example oxygen may be supplied by an air pump, the most efficient means for introducing oxygen into the liquid.

The enzyme or mixture of enzymes may also contain additional enzyme activities in addition to glucose oxidase. For example, the mixture may comprise catalase. Glucose oxidase and other enzymes that catalyze the oxidation of sugars to organic acids simultaneously catalyze the formation of H ₂ O ₂ . As described herein below, short exposure to H ₂ O ₂ can be beneficial to reduce the levels of amino acids, especially methionine, however, long-term exposure to large amounts of H ₂ O ₂ Exposure is undesirable and can cause unwanted oxidation. Thus, in embodiments of the invention where an enzyme capable of catalyzing the formation of acid and H ₂ O ₂ is then used, preferably the method also comprises incubation with catalase. The incubation with the catalase may be performed simultaneously with the incubation with an enzyme capable of catalyzing the formation of an acid, or may be performed in partial overlap or sequentially. The catalase may be any enzyme capable of catalyzing the decomposition of hydrogen peroxide to water and oxygen. Thus, catalase may be an enzyme classified under EC 1.11.1.6. In particular, catalase may be an enzyme that catalyzes the following reaction:
(Formula 1)
2H ₂ O ₂ → O ₂ + 2H ₂ O

  The catalase may be any useful catalase. For example, the catalase may be catalase of Aspergillus niger, Bacillus subtilis or bovine origin (in particular, bovine liver origin). It is generally preferred that no glucose isomerase be added to any of the liquids in the method of the invention. It is therefore preferred that no enzymes classified under EC 5.3.1.5 be added to any of the liquids in the method of the invention.

Functional Homologs The term "functional homologue" as used herein denotes a polypeptide which shares at least one biological function with a reference polypeptide. In general, said functional homologues also share significant sequence identity with the reference polypeptide.

  Preferably, a functional homologue of a reference polypeptide is a polypeptide having the same biological function as the reference protein and sharing a high level of sequence identity with the reference polypeptide.

  A high level of sequence identity indicates the possibility that the first sequence is derived from the second sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Thus, candidate sequences sharing 80% amino acid identity with the reference sequence require that, after alignment, 80% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. The identity according to the invention is not limited by computer analysis, such as, but not limited to, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson T., Thompson J. D., Higgins D. G., Gibson T. J. ., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. It is determined with the aid of the default parameters suggested in them. ClustalW software is available from the ClustalW WWW Service at the European Bioinformatics Institute http: // www. ebi. ac. Available at uk / clustalw. This program is used with its default settings to align the mature (bioactive) portion of the query and the reference polypeptide. The number of completely conserved residues is counted and divided by the length of the reference polypeptide. Thus, sequence identity is determined over the entire length of the reference polypeptide.

Increasing the Level of Acids by Microorganisms In one embodiment, the level of acid is increased with the help of microorganisms that can ferment sugars to form organic acids. In particular, the microorganism may be able to ferment glucose to form an organic acid. Such microorganisms are also referred to herein as "glucose-fermenting microorganisms". Preferably, said microorganism is capable of excreting said organic acid into the surrounding medium. In another embodiment of the invention, the level of base is increased with the aid of a microorganism capable of fermenting sugar to form a base.

  Preferably, the glucose-fermenting microorganism is a microorganism capable of converting glucose to an organic acid under anaerobic conditions. The organic acid may be any of the organic acids described herein above in the section "Reducing the Content of Amino Acids by Dialysis". In particular, the organic acid may be selected from the group consisting of lactic acid, citric acid, malic acid, tartaric acid, acetic acid, succinic acid, isocitric acid, α-ketoglutaric acid, fumaric acid and oxaloacetic acid. In a preferred embodiment, the organic acid is lactic acid.

  Thus, in a preferred embodiment of the invention, the glucose-fermenting microorganism can ferment glucose to obtain lactic acid. More preferably, the glucose-fermenting microorganism can absorb glucose, convert glucose to lactic acid under anaerobic conditions, and excrete at least some of said lactic acid.

  The glucose-fermenting microorganism used with the present invention may preferably be selected from the group consisting of yeast and bacteria. In particular, the glucose-fermenting microorganism may be a food grade microorganism, ie a microorganism that is acceptable for use in the manufacture of food and beverages for humans.

  In one embodiment, it is preferred that the glucose-fermenting microorganism is unable to grow to a considerable extent in beer, more preferably said microorganism is a microorganism unable to grow in beer. In particular, the microorganisms may be bacteria which can not grow in beer.

  It is even more preferred that the microorganism is completely devoid of extracellular proteases, ie the microorganism does not express and excrete proteases. In one embodiment, the microorganism is a yeast. The yeast is, for example, the Kleberomyces family, such as K. coli. Lactis or K. coli. It may be a Marxianus yeast. The yeast may also be any of the organic acid producing yeasts described in Loureiro V, Malfeito-Ferreira M: Spoilage yeasts in the wine industry, International Journal of Food Microbiology 2003: 86: 23-50. For example, the yeast may be of the Chloekera, Deckera / Bretanomyces or Pichia family.

  In one embodiment of the invention, the yeast may be selected from the group consisting of the yeasts listed in Table 1 of the International Patent Application PCT / DK2013 / 050215.

  In one embodiment of the invention, the glucose-fermenting microorganism is a lactic acid bacterium. The lactic acid bacteria may be, for example, bacteria of the order Lactobacillus. In particular, the lactic acid bacterium may be a bacterium of a genus selected from the group consisting of: Bifidobacterium, Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Aerococc, Carnobacterium, Enterococcus , Oenococcus, Sporolactobacillus, Streptococcus, Tetragenococcus, Bagococci and Waisera. In particular, the lactic acid bacterium may be a bacterium of a genus selected from the group consisting of Bifidobacterium, Lactobacillus, Lactococcus and Streptococcus.

  Thus, in one embodiment, the glucose-fermenting microorganism may be a Lactobacillus selected from the group consisting of: Jungangensis, L. Fugenensis, L. et al. Garvie, L. L. lactis, L. et al. Pythium, L. Plantarum and L. Raffinola lactis. Preferably, the glucose-fermenting microorganism may be Lactococcus lactis.

  Thus, in one embodiment, the glucose-fermenting microorganism is L.. Acetotolerance L. Asisifarine, L. L. acidosis. Acidophilus, L .; Aziris, L .; Algidas, L .; Alimentarius, L. Amylolytics, L. et al. Amylophilus, L. et al. Amilotrophicas, L. et al. Ami Roboras, L .; Animalis, L .; Entree, L. Apodemi, L. Aviarias, L. Bifermentans, L .; Brevis, L .; Buchneri, L .; Kamelie L. Kasei, L. Catena Holmis, L .; Seti, L. Cleohominis, L .; Corinoides, L. Compost, L. Concavas L. Colini Holmis, L .; Crispatas, L .; Krustrum, L. Kurvatus, L .; Del Brucky, L. Dextrinics, L. Diorolivance, L. et al. Echi, L. Eggenero, L. Farraginis, L. Falsiminis, L .; Fermentum, L. Hornikalis, L .; Fructivorans, L. et al. Fullmenty, L. Futensis, L .; Galinaram, L .; Gasseli, L. Gastrics, L. Ganensis, L .; Graminis, L .; Humeshi, L. Hamsteri, L. Harbinensis, L .; Hayakitensis, L .; Helvetics, L. Hilgardi, L. Homo Hochi, L. Inels, L .; Incubier, L .; Intestinalis, L .; Jensen, L .; John Soni, L. Calyxensis, L .; Kefiranofaciens, L. et al. Kefili, L .; Kimchi L. Kitasatnis, L. Kunkei, L. Reichmann, L. Lindneri, L .; Malefermences, L .; Mari, L. Manihothibirans, L .; Mindensis, L .; Mucosae L. Murinas, L .; Nagerry, L .; Namlensis, L .; Nantensis, L .; Oligofermence, L. et al. Oris, L. Panis, L .; Pantheris, L .; Parabrevis, L .; Parabhunelli L. Paracolyoides, L. Paracofaraginis L. Parakefili, L .; Paralimentarius, L. Paraplantarum, L .; Pentosus, L .; Perorence, L. Plantarum, L .; Pontis, L .; Shishishi, L. Lenini, L .; Reuteri, L .; Lamnosus, L .; Rime, L. Rogosse, L. Rossier, L .; Ruminis, L. Serimneri, L. Sakei, L. Salibarius, L. Satsumensis, L .; Secariphilus, L .; Charpe, L .; Silliginis, L .; Spicelli, L. Cevicus, L. Tyllandensis, L .; Wurtunensis, L .; Vaccinostera, L .; Vaginaris, L. Vers Moldensis, L. Vini, L. Vitrinas, L. Zee and L. It may be a Lactobacillus selected from the group consisting of Dimae, preferably, Amylolytics, L. et al. Del Brucky and L. It may be selected from the group consisting of fermentum.

  Thus, in one embodiment, the glucose-fermenting microorganism is P. Acidi Ractici, P .; Seri Cola, P.I. Clausseny, P .; Damnosus, P .; Dextrinics, P .; Ethanololidurans, P .; Inopinatas, P .; Palbulls, P .; Pentosaceus and P. a. It may be a Pediococcus selected from the group consisting of Stelesiais, preferably Pediococcus is preferably P. Acidi Ractici, P .; Dextrinics and P.W. It may be selected from the group consisting of Pentosaceus.

  In one embodiment, the glucose-fermenting microorganism may be Gluconobacter, such as Gluconobacter oxydans. Gluconobacter, particularly Gluconobacter oxydans, can ferment various sugars to form organic acids. Thus, for example, Gluconobacter, particularly Gluconobacter oxydans, can ferment various sugars, including maltose and glucose, to obtain gluconic acid. Thus, in embodiments of the invention where the glucose-fermenting microorganism is Gluconobacter, then step b) may be omitted from the method of the invention. Therefore, Gluconobacter is an example of a microorganism that can ferment sugar to form an organic acid.

  The cereal extract may be incubated with the glucose-fermenting microorganism in any suitable manner. In general, the incubation is carried out in a closed vessel or vessel. In one preferred embodiment, the incubation is performed in a tank connected to one or more chambers defined by two anion exchange membranes, as described herein below in section "REED".

  Incubation may be performed for any suitable time. In general, the incubation may be in the range of 12 hours to 1 week, such as 12 hours to 48 hours, such as 12 hours to 30 hours, such as 20 to 28 hours. In general, the incubation should be for a time sufficient to reduce the total level of Strecker amino acids to less than 100 mg / L, preferably less than 50 mg / L, for example less than 30 mg / L.

  Incubation may be performed at any suitable temperature. Preferably, the temperature is selected to be an appropriate temperature to allow growth of the particular glucose fermentation microorganism. Generally, the temperature is in the range of 15-40 ° C, such as in the range of 20-35 ° C, such as in the range of 23-32 ° C. This may be especially the case when the glucose-fermenting microorganism is a lactic acid bacterium, eg Lactococcus lactis.

REED
The present invention is for preparing a beverage comprising a method for reducing the content of one or more Strecker amino acids in cereal extract and a step of reducing the content of one or more Trekker amino acids in cereal extract On the way. Surprisingly, the inventors have found that one particularly efficient method for reducing the level of amino acids involves the following steps:
a) increasing the level of the acid or base in the cereal extract; and b) at least a portion of the acid anion of the increased acid or at least a portion of the basic cation of the increased base. Removing through a dialysis (REED) membrane stack.

  In this regard, it should be noted that REED treatment may not by itself be sufficient to remove amino acids. Thus, under some conditions, REED treatment alone may have little effect on the level of amino acids.

The term "REED" as used herein denotes a method in which ions are removed from the liquid with the aid of a membrane stack consisting of:
a) At least one cell consisting of:
1. 2. two ion exchange membranes defining a chamber for the liquid to be treated; Two additional chambers for dialysate, said two additional chambers being arranged on opposite sides adjacent to the chamber for the liquid to be treated, said two additional chambers being able to be connected b) a set of end membranes c) a means for applying an electric field on the membrane stack by at least two electrodes d) a means for reversing the direction of the electric field in the membrane stack, wherein the removal comprises the steps of including:
1. Inserting the liquid to be treated into a chamber for the liquid to be treated; Inserting dialysate into two additional chambers for dialysate; and Applying an electric field on the membrane stack; Incubating the liquid to be treated in the chamber; Reversing the direction of the electric field at intervals.

  In the method of the invention, the liquid to be treated is typically a cereal extract, such as any of the cereal extracts described herein above in section "Cereal Extract". In general, each REED membrane stack is composed of several membranes which define alternating chambers for the fluid to be processed and the dialysate. The cereal extract can be maintained in a tank, which is connected to the chamber (s) for the liquid to be processed and can be circulated through the REED membrane stack. Thus, the cereal extract can be maintained in the tank, and the level of acid or base can be increased in the tank for a predetermined time before the cereal extract is circulated through the REED membrane stack . The cereal extract may be circulated through the REED membrane stack for a predetermined time. It is also included within the invention that more than one REED membrane stack can be connected to a tank containing cereal extract. Preferably, the cereal extract can be circulated independently through each of the membrane stacks.

  Regardless of the direction of the electric field, ions could be moved from the chamber defining the liquid to be processed into any of the chambers for dialysate.

Acidic anions can be removed, in particular, through an anion exchange reverse electrical enhancement dialysis (AX-REED) membrane stack, said membrane stack comprising: a) at least one cell consisting of:
i. Two anion exchange membranes defining a chamber for the liquid to be treated; and ii. Two further chambers for dialysate, said two further chambers being arranged on the opposite side, adjacent to the chamber for the liquid to be treated, said two further chambers being able to be connected b) a set of end membranes c) a means for applying an electric field on the membrane stack by at least two electrodes d) a means for reversing the direction of the electric field in the membrane stack, wherein the removal comprises the steps of including:
1. Circulating the liquid from a tank containing cereal extract into a chamber for the liquid to be processed; Inserting alkaline dialysate into two additional chambers for dialysate; Applying an electric field on the membrane stack; Incubating the liquid in the chamber; Reverse the direction of the electric field at intervals; Circulating the treated liquid back to the tank.

  This step may also be referred to as AX-REED processing. Generally, AX-REED treatment is performed to maintain the pH constant or above a predetermined level. Thus, AX-REED is preferably adjusted as compared to the continuous increase in acid, so that the continuous decrease in pH associated with the increase in acid is counteracted by the removal of acid ions by AX-REED. The AX-REED treatment is adjusted to maintain the pH in the range of 3 to 7, preferably the pH is maintained in the range of 4 to 6, for example 4 to 5.5, for example at a pH of 6 or less It is preferred to be maintained. In another embodiment of the present invention, the pH is preferably maintained at a pH of at least 5, more preferably at a pH of at least 5.5, such as a pH of at least 6. Thus, in this embodiment, the pH is very preferably maintained in the range of 5.5-7. In an embodiment of the invention, the cereal extract is treated to increase the level of acid either by addition of said acid or use of an enzyme or mixture of enzymes capable of catalyzing the formation of said acid, AX-REED Is used to remove the acid and then the AX-REED treatment is adjusted to maintain the pH at less than 5.5 or at least 6, for example in the range of 6 to 7.5, for example in the range of 6 to 7 May be preferred. In an embodiment of the invention, the cereal extract is treated to increase the level of acid using a microorganism capable of excreting an organic acid, said acid being removed using AX-REED, so that AX- It may be preferred that the REED be adjusted to maintain the pH at a pH which is convenient for the microorganism, for example at a pH of at least 5.5, for example in the range of 5.5 to 7.5.

  It is included within the invention that after AX-REED treatment, the pH can be reduced, for example by an acidification step. Thus, AX-REED treatment can be adjusted to maintain the pH in the range indicated above, however, after AX-REED treatment the pH can be reduced so that the pH of the final beverage is Lower than the indicated range. For example, the treated cereal extract may be subjected to an acidification step after AX-REED treatment, which is carried out in any of the ways described hereinafter in the section "Combination Method". obtain.

  AX-REED treatment can also be continued, so that at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90% of the added and / or generated acidic ions are removed. Be done.

The basic cations can be removed, in particular, through a cation exchange reverse electrical enhancement dialysis (CX-REED) membrane stack, said membrane stack comprising:
a) At least one cell consisting of:
i. Two cation exchange membranes defining a chamber for the liquid to be treated; and ii. Two further chambers for a second dialysate, said two further chambers being arranged on opposite sides adjacent to a chamber for the liquid to be treated, said two further chambers being connected V) means for applying an electric field on the membrane stack by means of a chamber b) a set of end membranes c) at least two electrodes d) means for reversing the direction of the electric field in said membrane stack, wherein removal is: Includes the following steps:
1. Circulating the liquid from a tank containing cereal extract into a chamber for the liquid to be processed; 2. Inserting the acidic second dialysate into two additional chambers for the second dialysate; Applying an electric field on the membrane stack;
4. Incubating the liquid in the chamber;
5. Reverse the direction of the electric field at intervals; Circulating the treated liquid back to the tank.

Thus, the method for reducing the level of amino acids may include the following steps:
I. Inserting the cereal extract into a tank connected to the AX-REED membrane stack described above and the CX-REED membrane stack described above;
II. Increasing the level of acid in the cereal extract;
III. Removing at least a portion of the acid anions of the increased acid in cereal extracts by AX-REED treatment as described herein;
IV. Removing at least a portion of the cations in the cereal extract by CX-REED treatment as described herein.

Steps II, III and IV may be performed sequentially, partially simultaneously or simultaneously. Steps II, III and IV can also be independently repeated two more times. In a preferred embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Performing step II, thereby increasing the level of acid;
3. Performing steps II and III simultaneously, thereby increasing or maintaining the pH;
4. Performing steps II, III and IV simultaneously, thereby desalting;
5. Carrying out steps II and IV simultaneously, thereby acidifying or otherwise acidifying the liquid.

In another embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Performing step II, thereby increasing the level of acid;
3. Performing steps II and III simultaneously, thereby increasing or maintaining the pH;
4. Performing steps III and IV simultaneously, thereby desalting;
5. Performing step IV, thereby acidifying or otherwise acidifying the liquid;

In one embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Performing step II, thereby increasing the level of acid;
3. Performing step II and simultaneously increasing the level of base, thereby controlling pH;
4. Performing steps II and III simultaneously, thereby increasing or maintaining the pH;
5. Performing steps II, III and IV simultaneously, thereby desalting;
6. Carrying out steps II and IV simultaneously, thereby acidifying or otherwise acidifying the liquid.

In one embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Performing step II, thereby increasing the level of acid;
3. Performing step II and simultaneously increasing the level of base, thereby controlling pH;
4. Performing steps II and III simultaneously, thereby increasing or maintaining the pH;
5. Performing steps III and IV simultaneously, thereby desalting;
6. Performing step IV, thereby acidifying or otherwise acidifying the liquid;

  The acidifying step may also be obtained by a method other than performing step II and / or IV. Thus, instead of removing basic cations by CX-REED treatment, acidification can be obtained by simply adding an acid. Acidification is also obtained by increasing the level of acid, either in the manner described herein above in the section "Increasing the level of acid by enzymes" or "Increasing the level of acids by microorganisms". The obtained acidic anion is not removed by AX-REED. Acidification may be carried out, for example, to a pH in the range of 3 to 7, for example 4 to 6, until the desired acidity of the beverage is obtained. Useful methods for acidifying liquids are also described herein below in the section "Combination Methods".

Alternatively, the method for reducing the level of amino acids may comprise the following steps:
I. Inserting the cereal extract into a tank connected to the AX-REED membrane stack described above and the CX-REED membrane stack described above;
II. Increasing the level of base in the cereal extract;
III. Removing at least a portion of the basic cations of the increased base in cereal extracts by the CX-REED treatment described herein;
IV. Removing at least a portion of the anions in the cereal extract by AX-REED treatment as described herein.

Steps II, III and IV may be performed sequentially, partially simultaneously or simultaneously. Steps II, III and IV can also be independently repeated two more times. In a preferred embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Carrying out step II, thereby increasing the level of base;
3. Performing steps II and III simultaneously, thereby reducing or maintaining the pH;
4. Performing steps II, III and IV simultaneously, thereby desalting;
5. Carrying out steps II and IV simultaneously, thereby alkalizing.

In another embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Carrying out step II, thereby increasing the level of base;
3. Performing steps II and III simultaneously, thereby reducing or maintaining the pH;
4. Performing steps III and IV simultaneously, thereby desalting;
5. Process IV is carried out and alkalized thereby.

In one embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Carrying out step II, thereby increasing the level of base;
3. Performing step II and simultaneously increasing the level of acid, thereby controlling pH;
4. Performing steps II and III simultaneously, thereby reducing or maintaining the pH;
5. Performing steps II, III and IV simultaneously, thereby desalting;
6. Carrying out steps II and IV simultaneously, thereby alkalizing.

In one embodiment, the method comprises the following steps performed in the order shown:
1. Performing step I;
2. Carrying out step II, thereby increasing the level of base;
3. Performing step II and simultaneously increasing the level of acid, thereby controlling pH;
4. Performing steps II and III simultaneously, thereby reducing or maintaining the pH;
5. Performing steps III and IV simultaneously, thereby desalting;
6. Process IV is carried out and alkalized thereby.

  The tank contains cereal extract at the start of the procedure, but after a while, the tank partly contains AX-REED treated liquid and / or partly CX-REED treated liquid, which also may be acid or It is processed to increase the level of the base. For the sake of simplicity, the tank may nevertheless be referred to as a "tank containing cereal extract".

  AX-REED treatment and / or CX-REED treatment may be performed using REED equipment. The REED installation according to the invention preferably comprises at least one AX-REED membrane stack and at least one CX-REED membrane stack, which is any of the AX-REED membrane stacks described herein in this section. And any of the CX-REED membrane stacks described herein in this section. Even more preferably, the REED installation comprises at least one AX-REED and at least one CX-REED membrane stack, wherein said AX-REED and said CX-REED membrane stack are connected in parallel, both cereals Connected to the tank containing the extract. Thus, the REED facility can include one AX-REED membrane stack and one CX-REED membrane stack connected in parallel.

  When two or more REED stacks are arranged in parallel, the processed liquid, ie the processed liquid from one REED stack, is directly following the REED stack, as in the case where two stacks are connected in series. Not induced to Rather, the liquid is returned to the tank.

  The parallel system can, for example, have AX-REED and CX-REED connected to a reservoir and / or tank containing cereal extract. The AX-REED receives the liquid to be processed from the reservoir and / or tank, and the liquid to be processed is returned to the reservoir and / or system after being processed by the AX-REED stack. At the same time or another time, the liquid to be processed by the CX-REED is received from the reservoir and / or the tank, and the liquid is returned to the reservoir and / or the tank after being processed by the CX-REED stack. It is understood that liquid can be recirculated from the tank to the AX-REED and / or CX-REED stacks.

  Alternatively, the REED facility may include more AX-REED membrane stacks than the CX-REED stack, or the REED facility may include more CX-REED membrane stacks than the AX-REED stack. The relative number of AX-REED membrane stack / CX-REED membrane stack is how much of the first component is removed from the liquid versus how much of the second component is removed from the liquid Can be varied to control. The ratio between the first component to be removed and the second component to be removed may also be adjusted by providing different sized AX-REED membrane stacks and a CX-REED membrane stack.

  The REED stack comprises at least one chamber for the liquid to be processed and at least two chambers for the dialysate. The chamber (s) containing the liquid to be processed and the chambers containing the dialysate are arranged alternately, ie the REED stack comprises at least three active adjacent chambers: chambers for the dialysate-treated Chamber for liquid-Chamber for dialysate. Each interface between the chamber for the liquid to be processed and the chamber for the dialysate is formed by an ion exchange membrane, this exchange membrane in the AX-REED stack is an anion exchange membrane and in the CX-REED stack , Cation exchange membrane.

  Each REED stack also includes two end membranes that define an electrode chamber at each end of the REED stack, ie, a REED stack having two end membranes includes at least five adjacent chambers: electrode chamber-dialysate Chamber-Chamber for the liquid to be treated-Chamber for the dialysate-Electrode chamber.

  Each electrode chamber can be formed by the end membrane and the end wall of the REED stack.

In a REED stack with seven adjacent chambers, two electrode chambers and five active chambers are arranged as follows: electrode chamber-chamber for dialysate-chamber for the liquid to be treated-dialysate Chamber for-Chamber for the liquid to be treated-Chamber for the dialysate-Electrode chamber. Similarly, a REED stack with another odd number of adjacent chambers is arranged as follows: electrode chamber-chamber for dialysate-[chamber for liquid to be treated-chamber for dialysate _N -electrode chamber, where n is an integer, such as an integer in the range of 1 to 500, such as 2 to 200, such as 2 to 50, such as 2 to 25;

  FIG. 8 shows an exemplary REED installation 1 to be used with the method according to the invention, said REED installation comprising an AX-REED stack 2 arranged parallel to the CX-REED stack 3. Both AX-REED and CX-REED stacks are connected by piping 5 to tank 4 containing cereals extract and to the REED stacks to provide dialysate from which to derive dialysate fluid systems 6a and 6b . Fluid system 6a is for providing dialysate for use with AX-REED, while fluid system 6b is for providing a second dialysate. At the beginning of the process, tank 4 contains cereal extract and later, the tank partly contains AX-REED and / or CX-REED treated liquid. At the end of the process, tank 4 contains processed cereal extract.

The AX-REED stack is arranged to provide an electric field in five active chambers between the electrodes, ie alternating chambers containing the dialysate 9 formed by the membrane and the liquid 10 to be treated 2 electrodes 8 are included. In the present exemplary stack, alternating chambers are formed by:
An end membrane 11a defining a first electrode chamber 7a on one side and a first chamber 9 for dialysate on the opposite side
A first anion exchange membrane 12a defining a first chamber for the dialysate together with the first end membrane
A second anion exchange membrane 12b which forms a second chamber 10a for the liquid to be treated together with one anion exchange membrane
· A third anion exchange membrane 12c which forms a third chamber 9b for the dialysate together with the second anion exchange membrane
A fourth anion exchange membrane 12d which, together with the third anion exchange membrane, forms a fourth chamber 10b for the liquid to be treated
-A second end membrane 11b which forms a fifth chamber 9c for the dialysate together with a fourth anion exchange membrane
The first and second electrodes are arranged in the first electrode chamber 7a and the second electrode chamber 8a, respectively. The first electrode chamber is defined by a first end wall (indicated by a dotted line) and a first end membrane, and the second electrode chamber is indicated by a second end wall (also indicated by a dotted line). And a second end membrane.

  The exchange membranes 12a-12d can preferably be of the same type, as well as the two end membranes can also be identical.

  Similarly, the CX-REED stack comprises two electrodes 13 and 14 on either side of the stack of membranes, said stack of membranes comprising a first end membrane 15a, four cation exchange membranes 16a-16d and a second end membrane 15b. It is. Said membrane together with the end wall, a first electrode chamber 13a, a first chamber 17a for dialysate, a first chamber 18a for fluid to be treated, a second chamber 17b for dialysate, treatment Form a second chamber 18b for the fluid to be treated, a third chamber 17c for the dialysate and a second electrode chamber 14a.

  In this example, the dialysate may be any of the dialysate used with AX-REED described in this section, and the second dialysate is the second dialysate described in this section. It may be either.

  The REED facility can also include more than one AX-REED membrane stack connected in series, wherein the AX-REED membrane stack is connected in parallel to at least one CX-REED membrane stack. The REED facility can also include more than one AX-REED membrane stack connected in series, and more than one CX-REED membrane stack connected in series, where the AX-membrane stack and the CX- The REED membrane stacks are connected parallel to one another. Less preferably, the REED facility can also include AX-REED and CX-REED membrane stacks connected in series, where one of the stacks through its inlet to the reservoir and / or tank While the other is connected to the tank via its outlet.

  Each AX-REED membrane stack may comprise more than one cell identified above. For example, each AX-REED membrane stack may comprise in the range of 2 to 500 cells, such as in the range of 2 to 200 cells, such as in the range of 2 to 25 cells.

Removal of acidic ions typically involves the following steps:
1. Inserting the liquid to be treated into a chamber for the liquid to be treated; Inserting dialysate into two additional chambers for dialysate; and Applying an electric field on the membrane stack; Incubating the liquid to be treated in the chamber; Reversing the direction of the electric field at intervals.

  AX-REED can be carried out under circulation, and after incubation of the liquid to be treated in said chamber, the obtained liquid is removed from the chamber and then another chamber for the liquid to be treated or Furthermore, it is meant to be inserted into the same chamber for the liquid to be processed. If inserted into the same chamber, then frequently the dialysate in the two additional chambers was replaced with fresh dialysate.

  If more than one membrane stack is used, the liquid to be treated can be separately inserted into each of the chambers for the liquid to be treated. Alternatively, some or all of the chambers may be connected and so may be simultaneously supplied to some or all of the chambers. Similarly, dialysate may be separately inserted into each of the chambers for dialysate. Alternatively, some or all of the chambers may be connected so that some or all of the chambers are supplied simultaneously.

  The acidic ion to be removed may, for example, be the anion of any organic acid, such as any anion of the organic acids described herein above in the section "Reduction of the content of amino acids by dialysis". Preferably, at least the acid anions which are increased by addition and / or by production with the aid of enzymes or microorganisms are removed by AX-REED treatment.

Interestingly, as disclosed by the present invention, Strecker amino acids are removed even under such conditions. Amino acids contain both acidic -COOH group and alkaline -NH ₂ group. The few amino acids also contain other charged groups, but all Strecker amino acids are nonpolar amino acids with nonpolar side chains. This is also reflected by the pi of all Strecker amino acids, which is near neutral, in the range of about 5.5-6. Interestingly, even at lower pH, amino acids are still effectively reduced by the methods described herein. Thus, in one embodiment of the invention, the REED treatment is such that the pH is maintained in the range of 3 to 7, for example 4 to 6, for example 4 to 5.5, for example 6 or less throughout the REED treatment To be implemented.

  During the removal of acid ions, the two membranes surrounding the chamber for the liquid to be treated promote the transport of ions either from the liquid to be treated or from the dialysate into the liquid to be treated.

  The direction of the electric field is changed at intervals. Each reversal of the direction of the electric field results in a short-term reconstruction of the polarization profile of the affected ions on the surface and inside of the membrane, since the two membranes surrounding each feed compartment exchange function. This causes a short-term reversal of the separation process, since previously removed ions are pushed back into the feed solution until the membrane profile is rebuilt. It is advantageous to maintain the spacing between current reversals in any one REED stack, as permitted by fouling build-up, since each reversal causes short separation interruptions, small process instability Is introduced.

  Methods of the invention may include the use of more than one AX-REED membrane stack. The membrane stacks can be stacked on top of each other or side by side (usually separated by membrane spacers) until a sufficient membrane separation area is achieved. For feasible handling, operation and maintenance purposes, the membrane stack can be operated with several separate, practical sized membrane stacks, each with its own set of flow connections and electrodes Have the same separation function. These stacks are operated together as part of the same separation system, in parallel or in series, or some combination thereof. When more than one set of electrodes is used, it is advantageous to operate with multiple AX-REED membrane stacks. Thus, the number of AX-REED membrane stacks can vary from 2 to several hundred depending on the process in question, but typically the range of 2-50 AX-REED membrane stacks, more typically 4-20. It is a range of membrane stacks.

The dialysate used with AX-REED according to the invention may be any alkaline solution. Typically, it is an aqueous solution of a cation -OH, wherein said cation may be typically a metal cation. For example, the dialysate is selected from the group consisting of Ca (OH) ₂ , Mg (OH) ₂ , KOH and NaOH, preferably selected from the group consisting of Ca (OH) ₂ , Mg (OH) ₂ and KOH It may contain one or more bases. The dialysate typically comprises 0.01 to 30%, preferably 0.01 to 20%, more preferably 0.01 to 150% of the base, for example 0.01 to 10%. At concentrations in the range of In certain embodiments, the base is used at a concentration ranging from 0.01 to 6%. This can be especially true if the dialysate is used only once. All percentages are provided as w / w.

  In the case of AX-REED, acid ions are extracted through the anion exchange membrane in each chamber of the AX-REED membrane stack while hydroxide ions typically enter through the opposite anion exchange membrane. When the direction of the electric field is reversed, the extracted acid ions inside the first mentioned membrane are pushed back to the liquid to be treated and then the hydroxide ions start to enter the liquid to be treated. Thus, within a short period of time, pH control is not observed until the hydroxide profile has been rebuilt through the membrane previously used to extract the acidic ions. The length of the time phase after each current reversal until pH control is regenerated depends on various process conditions and membrane characteristics; typically, 30 to 180 until the process operates again with optimal process parameter control. It takes seconds. This is detected as a sudden change in process parameters, such as pH, which must then be controlled to return to the desired set point. Reversal of the electric field on each separate stack is preferably performed non-synchronously in order to widen the destabilizing effect and reduce the overall effect of current reversal with more than one membrane stack. Thus, in the invention, preferably more than one AX-REED membrane stack is used, and the electric field on each separate stack is non-cosynchronously reversed. The spacing for storing the electric field of each stack is typically of similar length, but the timing of the inversion is dispersed for the best process stabilization effect.

  In one embodiment of the invention, the orientation of the electric field in any first membrane stack is reversed at substantially periodic dispersion intervals relative to the reversal for any second or additional membrane stacks. .

  The spacing length between current reversals for one stack is typically selected for membrane fouling accumulation. Typically, said spacing in any one REED stack is in the range 5-6000 seconds, preferably 8-3000 seconds, more preferably 10-2000 seconds, even more preferably 100-1500 seconds. Can. In another embodiment of the invention, the orientation of the electric field in any first membrane stack is the time between current reversal of any first REED stack and any second or further REED stack in the same process. In order to maximize H.sub.2O.sub.2, inversion is performed at dispersion intervals of substantially equal length against inversion for any second or additional membrane stacks. With the same dispersion interval length between current reversals, ie if these reversals are uniformly distributed, connected reservoirs and / or tanks will experience reduced effects even more often. I will.

  In one embodiment of the invention, the strength of the applied electric field is adjusted according to the pH, salt concentration or conductivity of the liquid to be treated. By increasing the strength of the electric field, ion exchange is increased in the REED system, and vice versa. On-line, semi-on-line (e.g. time delay) or secondary (e.g. using on-line conductivity or turbidity measurements to estimate target ion concentration) measurements of controlled process parameters are control control mechanisms, e.g. It is input in the PID-control software, which in turn controls the output of the power supply to the REED electrode.

  Current reversal is not the only effect, which can introduce deviations in process control. It may be advantageous to control the concentration and flow and temperature of the various ions in the dialysate and the mode of operation for optimum control of the process parameters. With respect to temperature, then, the temperature in the reservoir and / or tank is typically selected to allow for microbial growth or high activity of the enzyme.

  If multiple stacks are used, it is possible to set the dialysate flow with or without booster pumps between stacks in either parallel or serial mode, as well as the liquid to be processed.

  In embodiments of the invention where the level of acid is increased, then the anion exchange REED (AX-REED) generally functions to exchange the generated organic acid with hydroxide ion, thus pH at the acid formation. Counter the reduction. By control of AX-REED, hydroxide exchange can maintain pH during fermentation without the need for neutralizing agent addition.

  The terms "reversal of the electric field" or "current reversal" in the context of the present invention mean a change of polarity of the REED electrode, resulting in a reversal of the direction of the current DC, whereby the migration of ions through the ion exchange membrane Is promoted.

  The anion exchange membrane may be any useful anion exchange membrane. The size of the membrane can be selected to achieve a suitable membrane area as compared to the volume of cereal extract being processed. Non-limiting examples of useful anion exchange membranes include Ionic AR103 (GE, USA), Neosepta ASM (Astom Corp., Japan), Fumatech FAB (Fumatech, Germany).

  Non-limiting examples of useful methods and equipment for carrying out AX-REED are also described in European Patent Applications EP 13478823, EP 2349541 and EP 2349540, all incorporated herein by reference.

  In some embodiments, AX-REED processing is performed first, and then AX-REED and CX-REED are performed simultaneously in parallel. In another embodiment, CX-REED treatment is performed first, and then AX-REED and CX-REED are performed simultaneously in parallel. AX-REED treatment typically leads to deacidification, CX-REED treatment typically leads to acidification, where simultaneous execution of AX-REED and CX-REED results in desalting of the liquid to be treated Lead to

  Regardless of the direction of the electric field, ions could be transferred from the chamber for the liquid to be processed into one of the chambers for the second dialysate.

  Each CX-REED membrane stack may comprise more than one cell identified above. For example, each CX-REED membrane stack may comprise cells in the range of 2 to 500, such as cells in the range of 2 to 200, such as in the range of 2 to 50, such as in the range of 2 to 25.

  The CX-REED can be carried out in circulation, and after incubation of the liquid to be treated in said chamber, the liquid obtained is removed from the chamber and then another chamber for the liquid to be treated or further Is meant to be inserted into the same chamber. If inserted into the same chamber, then frequently the second dialysate in two adjacent additional chambers was replaced with a fresh second dialysate.

  The cation to be removed may be any cation. In embodiments of the invention in which AX-REED treatment is first performed alone, it may then be one or more cations introduced into the fluid from the dialysate during AX-REED treatment. Thus, the cation may be, for example, any of the cations of the bases contained in the dialysate described hereinbefore. In embodiments of the invention where the level of base is increased, then the cation may in particular be the basic cation of said increased base.

  During the removal of the cations, the two membranes surrounding the chamber for the liquid to be treated either transport ions from the liquid to be treated or transport ions from the second dialysate into the liquid Facilitate.

  The direction of the electric field is changed at intervals in the same manner as described herein above for AX-REED.

  Methods of the invention may include the use of more than one CX-REED membrane stack. The membrane stacks can be stacked on top of one another or side by side, sufficient membrane separation area being achieved and stacked (usually separated by membrane spacers) until the desired capacity is obtained. For feasible handling, operation and maintenance purposes, the membrane stack can be operated with several separate, practical sized membrane stacks, each with its own set of flow connections and electrodes Have the same separation function. These stacks are operated together as part of the same separation system, in parallel or in series, or some combination thereof. When more than one set of electrodes is used, it is advantageous to operate with multiple CX-REED membrane stacks. Thus, the number of CX-REED membrane stacks can vary from 2 to several hundred depending on the process in question, but typically the range of 2-50 CX-REED membrane stacks, more typically 4 -20 membrane stack range.

The second dialysate used with the CX-REED according to the invention may be any acidic solution. Typically, it is an aqueous solution of H-anions, where the anion is typically an inorganic anion. Thus, for example, the second dialysate may comprise one or more acids selected from the group consisting of H ₃ PO ₄ , HNO ₃ and H ₂ SO ₄ . Preferably, the second dialysate comprises H ₃ PO ₄ . The second dialysate typically contains the acid in the range of 0.01 to 30%, preferably in the range of 0.01 to 20%, more preferably in the range of 0.01 to 10%, for example 0.01 Including at concentrations ranging from ~ 6%. The percentages are provided as w / w.

In the case of CX-REED, cations are extracted through one cation exchange membrane of each cell of the CX-REED membrane stack (s), while typically H ⁺ ions enter through the opposite cation exchange membrane. When the direction of the electric field is reversed, the extracted cations inside the first mentioned membrane are pushed back to the liquid to be treated and then the H ⁺ ions begin to enter the liquid to be treated. Reversal of the electric field on each separate stack is preferably performed nonsynchronously in order to widen the destabilizing effect and reduce the overall effect of current reversal with more than one membrane stack. Thus, in the invention, preferably more than one CX-REED membrane stack is used, and the electric field on each separate stack is non-synchronously reversed. Even though the spacing for storing the electric field of each stack is typically of similar length, the timing of the inversion is distributed for the best process stabilization effect.

  In one embodiment of the invention, the orientation of the electric field in any first membrane stack is reversed at substantially periodic dispersion intervals relative to the reversal for any second or additional membrane stacks. .

  The spacing length between current reversals for one stack is typically selected for membrane fouling accumulation. Typically, said spacing in any one CX-REED stack is in the range 5-6000 seconds, preferably 8-3000 seconds, more preferably 10-2000 seconds and even more preferably 100-1500 seconds. May be

  In another embodiment of the present invention, the orientation of the electric field in any first membrane stack is current reversal of any first CX-REED stack and any second or further CX-REED stack in the same process. In order to maximize the time between, the inversion intervals are of substantially equal length to the inversion for any second or further membrane stack. With the same dispersion interval length between current reversals, ie if these reversals are uniformly distributed, coupled bioreactors will experience reduced effects even more often.

  In one embodiment of the invention, the strength of the applied electric field is adjusted according to the pH, salt concentration or conductivity of the liquid composition. By increasing the strength of the electric field, ion exchange is increased in the CX-REED system and vice versa. On-line, semi-on-line (e.g. time delay) or secondary (e.g. using on-line conductivity or turbidity measurements to estimate target ion concentration) measurements of controlled process parameters are control control mechanisms, e.g. It is input in the PID-control software, which in turn controls the output of the power supply to the CX-REED electrode.

  Reversal of the electric field is not the only effect, which can introduce deviations in process control. It may be advantageous to control the concentration and flow and temperature and mode of operation of the various ions in the second dialysate for optimal control of the process parameters.

  If multiple stacks are used, it is possible to set the flow of the second dialysate, with or without booster pumps between the stacks, in either parallel or serial mode.

  The cation exchange membrane may be any useful cation exchange membrane. The size of the membrane can be selected to achieve a suitable retention time. To calculate the retention time, the total area of the anion membrane used is taken into account. Thus, if the method employs the use of many membrane stacks and / or if each membrane stack includes many cells, then the area of each membrane may be reduced.

  Non-limiting examples of useful CX-membranes include Nafion N117 (Dupont) and Neosepta CMB (Astom Corp., Japan).

  Non-limiting examples of useful methods and equipment for carrying out AX-REED are also described in European Patent Applications EP 13478823, EP 2349541 and EP 2349540, all incorporated herein by reference.

  In general, cation exchange REED (CX-REED) functions to exchange cations with hydrogen ions. Thus, if AX-REED and CX-REED are operated simultaneously, then the cations are exchanged with hydrogen ions and the anions are exchanged with hydroxide ions. Hydrogen ions and hydroxide ions transported into the liquid can together form water and desalting occurs. Thus, by operating AX-REED and CX-REED simultaneously, the conductivity can be reduced. Preferably, the AX-REED treatment and the CX-REED treatment are carried out simultaneously for a time sufficient to obtain a treated cereal extract having a suitable conductivity. The conductivity is preferably at most 7 mS / cm, preferably at most 6 mS / cm, still more preferably at most 5 mS / cm, such as in the range of 1 to 5 mS / cm, such as in the range of 1 to 5 mS / cm, such as 1 to 4. The range is 5 mS / cm, for example about 4.5. If the liquid has a higher conductivity, then simultaneous operation of AX-REED and CX-REED can be continued until the liquid has the desired conductivity. In general, conductivities higher than 5 mS / cm are undesirable in processed cereal extracts, as this can cause salty taste.

Reducing the Content of Amino Acids by an Oxidant The method of the invention comprises the step of reducing the content of one or more amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine.

  In one embodiment, the step comprises incubating the cereal extract with an oxidizing agent. This is particularly appropriate in embodiments of the invention where the step of reducing the content of amino acids is a step of reducing the content of methionine.

The oxidizing agent may be any useful oxidizing agent. It is important that oxidizing agents can be used for the preparation of the beverage. For example, the oxidizing agent may be selected from the group consisting of peroxide and ozone (O ₃ ). Non-limiting examples of useful peroxides include H ₂ O ₂ and peracetic acid.

It may be advantageous to use an oxidizing agent that can be inactivated. This allows better control of the reaction. For example, peroxides such as H ₂ O ₂ are useful because they can be inactivated, for example by catalase. In particular, the oxidizing agent is preferably an inorganic peroxide. In particular, the oxidizing agent is preferably H ₂ O ₂ .

  The cereal extract is preferably incubated under conditions which result in a reduction of the content of methionine in the treated cereal extract for a period of time, so that said treated cereal extract is at most 15 mg / L, for example at most 10 mg / l. L, for example at most 5 mg / L, for example at most 3 mg / L of methionine.

For example, said cereal extract is at least 20 ppm H ₂ O ₂ , such as at least 40 ppm H ₂ O ₂ , such as at least 50 ppm H ₂ O ₂ , such as at least 100 ppm H ₂ O ₂ , such as at least 250 ppm H ₂ O _{2 2,} for example, of _H 2 _{O 2} ranging in 20～10000Ppm, for example, of _H 2 _{O 2} ranging in 20～5000Ppm, for example, of _H 2 _{O 2} ranging in 20～2500Ppm, for example, of _H 2 _O 2 ranges of 50 to 10000 ppm, for example of _H 2 _{O 2} ranges of 50 to 5000 ppm, for example, of _H 2 _{O 2} ranging in _{50～2500Ppm,} for example, of _H 2 _{O 2} ranging in 250～10000Ppm, for example in the range of 250~5000ppm _{H 2 O} 2, for example 250 _H 2 _{O 2} and incubator ranging ~2500ppm It may be over bet. The incubation may be between 1 and 30 hours. In embodiments where the level of H ₂ O ₂ is at least 100 ppm, such as at least 250 ppm, then the incubation is preferably between 1 and 10 hours, such as between 4 and 5 hours.

In contrast to the prior art belief that oxidation is detrimental to the taste of the beverage, the present invention is interestingly disclosing that oxidation can be an advantage. In one embodiment of the invention, the oxidizing agent can not be added directly to the cereal extract, but instead an enzyme is added, which can catalyze the formation of the oxidizing agent. Thus, it is included within the invention that the content of Strecker amino acids, in particular the content of methionine, can be reduced by incubation with an enzyme or mixture of enzymes capable of catalyzing the formation of H ₂ O _{2 in} cereal extracts. Be The enzyme capable of catalyzing the formation of H ₂ O ₂ may be any useful enzyme. For example, it may be any of the glucose oxidases described herein above in the section "Increased levels of acid by enzymes".

Glucose oxidase catalyzes the formation of both acid and H ₂ O ₂ , as described herein above in the section “Energy level of acid by enzymes”, and thus typically H ₂ O ₂ is The method formed with the aid of glucose oxidase comprises removing at least part of the acidic anions in the cereal extract by AX-REED treatment as described hereinabove. In embodiments of the invention, it may be preferable that the treated cereal extract not be subjected to subsequent alcohol fermentation, in which case the acidic anion is not removed by AX-REED.

However, in general, wort treated with an oxidizing agent such as H ₂ O ₂ can be further processed into a beverage, for example the wort may be subjected to conventional alcoholic fermentation. Thus, the beverage may be a beer brewed with wort, which is treated with an oxidizing agent rather than with standard wort.

Thus, in one aspect, the invention relates to a method for producing a flavor-stable cereal-based beverage, said method comprising the following steps:
i) providing a cereal extract comprising at least 25 mg / L methionine;
ii) treating the cereal extract with an oxidizing agent (eg H ₂ O ₂ ) to reduce the content of methionine, thereby obtaining a treated cereal extract;
iii) Process the processed cereal extract into a beverage, for example by fermentation, wherein the beverage has a content of methionine of less than 5 mg / L.

Combination Methods Combinations of various methods for reducing the level of amino acids described herein are also included within the invention.

Thus, in one embodiment of the invention, the present invention relates to a method for producing a flavor-stable cereal-based beverage, said method comprising the steps of:
i) providing a cereal extract comprising at least 25 mg / L methionine, such as any of the cereal extracts described herein above in section "Cericle extract";
ii) inserting the cereal extract into a tank connected to the AX-REED membrane stack described above and the CX-REED membrane stack described above;
iii) increasing the level of acid in the cereal extract using a microorganism capable of fermenting the sugar to form an organic acid, wherein the microorganism is treated with A process of any of the microorganisms described in "the increase in levels";
iv) removing at least a portion of the acid anions of said increased acid in cereal extract by AX-REED treatment described herein while maintaining a pH in the range of 5.5-7;
v) acidifying the liquid by one of the methods described below;
vi) thereby obtaining a processed cereal extract;
vii) Optionally further processing the processed cereal extract into a beverage, for example as described hereinafter in the section "Processing into beverages".

  The method may also include the step of desalting. The step of desalting is, for example, removing at least a portion of the acidic anions in the cereal extract by AX-REED treatment, and at least a portion of the cations in the cereal extract described hereinabove. Removal by treatment with CX-REED. In particular, the AX-REED and the CX-REED processes may be performed simultaneously. The step of desalting may preferably be carried out after step iv), more preferably between steps iv) and v).

  The acidification step may be performed in any useful manner. In one embodiment, acidification is obtained by removing basic cations by CX-treatment as described herein. The CX-REED treatment may be carried out until the desired acidic pH, for example a pH in the range of 3 to 7, for example a pH in the range of 4 to 6, is achieved. In such embodiments, it is preferred that the method comprises the step of desalting between steps iv) and v). Acidification can also be obtained by continuing to ferment the microorganism without the action of either AX-REED or CX-REED until the desired pH is achieved. Bacteria continuously produce organic acids, so that if the acid is not removed by AX-REED, the liquid will become more acidic continuously. In such inventive embodiments, the desalting step may not be suitable. Acidification may also be obtained by treatment with an enzyme or mixture of enzymes capable of catalyzing the formation of an acid. The enzyme or mixture of enzymes may be any one of the enzymes / mixtures described herein above in the section "Increasing levels of acid by enzymes". If acidification is performed with the aid of an enzyme, the liquid may be treated to remove the microorganisms prior to incubation with the enzyme. Since the enzyme produces organic acid continuously, then the liquid becomes more acidic continuously, unless the acid is removed by AX-REED. In such inventive embodiments, the desalting step may not be suitable.

The method may also include the additional step of treating the treated cereal extract with an oxidizing agent, which may be any of the oxidizing agents described herein above in Section “Oxidizing Agents”. This may be particularly appropriate in embodiments of the invention where acidification is performed using CX-treatment or using continuous fermentation. In the embodiment of the invention where the acidification is carried out using an enzyme or mixture of enzymes capable of catalyzing the formation of acid, then frequently said enzyme also catalyzes the formation of H ₂ O ₂ and this Can be enough. Said step of treatment with an oxidizing agent may preferably be carried out after step v), for example between step v) and vi).

Process to Beverage The invention provides a method for producing a beverage. As soon as a treated cereal extract having a reduced content of Strecker amino acids is prepared, it can then be processed into a beverage. The processed cereal extract is itself a beverage, however in most cases it is included within the invention that additional processing steps are required to reach the final beverage.

  In one embodiment, the processed cereal extract is subjected to a fermentation process. This is especially so in embodiments of the invention where the cereal extract still has a total content of at most 300 mg / L, more preferably at most 250 mg / L amino acids, methionine, phenylalanine, valine, leucine and isoleucine.

  Thus, the cereal extract may be subjected to conventional fermentation similar to that used for the preparation of beer. Those skilled in the art are familiar with useful fermentation methods used in the preparation of beer. Briefly, fermentation may include incubating the treated cereal extract with the microorganism for a predetermined time. Incubation is generally performed under anaerobic conditions.

  The microorganism is preferably a microorganism capable of fermenting sugar to alcohol. Thus, the preferred microorganism used for the fermentation of the processed cereal extract is a yeast, more preferably a yeast capable of producing ethanol. In particular, it is preferred that the yeast be able to ferment sugars (eg glucose and / or maltose) to obtain ethanol. Useful yeasts include brewer's yeast such as the yeast Saccharomyces cerevisiae or Saccharomyces pastorianus, the former being S. aureus. Known as Karlsbergensis.

  It is also included within the invention that the processed cereal extract can be processed before being subjected to fermentation. For example, one or more additional compounds, such as any of the additional compounds described herein below in Section "Additional Compounds" may be added to the treated cereal extract. The processed cereal extract can also be diluted with water, for example, to obtain the desired content of sugar prior to fermentation. The treated cereal extract may also be mixed with one or more liquids, for example, the treated cereal extract may be mixed with another cereal extract. This results in a combined cereal extract containing reduced levels of one or more Strecker amino acids, which may be useful as a starting material for fermentation.

  After completion of the fermentation, the fermented processed cereal extract may be the final beverage. However, additional processing, such as filtration, cooling or carbonization may also be performed. One or more additional compounds, such as any of the additional compounds mentioned below in the section "Additional Compounds" may also be added to the beverage after fermentation.

  In another embodiment of the invention, the beverage is not subjected to fermentation by an alcohol producing yeast strain. Thus, in some embodiments of the invention, the beverage may preferably be a non-alcoholic beverage.

  The step iii) of processing the processed cereal extract into a beverage may also comprise or consist of conventional processing steps, such as filtration, cooling and / or carbonization. One or more additional compounds may also be added to the processed cereal extract, for example any of the additional compounds mentioned in the section "Additional compounds" in the following. Furthermore, the processed cereal extract may be mixed with one or more other liquids, such as another beverage, to obtain a mixed beverage.

Additional Compounds The method of the invention may include the step of adding one or more additional compound (s). The additional compound may be, for example, a flavor compound, a preservative or a functional ingredient component.

  The flavoring compound may be any of the flavoring compounds described herein below in section "Flavor compounds".

  The functional material component may be any material component added to obtain a certain function. Preferably, the functional ingredient component makes the beverage healthy. Non-limiting examples of functional ingredient components include water soluble fibers, proteins, added vitamins or minerals.

  The preservative may be any food grade preservative, for example it may be benzoic acid, sorbic acid, sulfite and / or their salts.

Additional compounds may also be CO _2. In particular, CO ₂ can be added to obtain carbonated beverages.

  The flavor compound used with the present invention may be any useful flavor compound. The flavor compound may be selected from the group consisting of, for example, aroma, plant extract, plant concentrate, plant parts and herbal dips.

  Thus, the flavor compound may, for example, be aromatic. The aromas are typically organic compounds, for example they may be plant secondary metabolites. The aroma may be any aroma, for example fruit aroma or vanilla aroma.

  The plant extract may, for example, be a herbal extract. Non-limiting examples of herbal extracts include extracts of green tea, black tea, rooibos, peppermint or hops. Plant extracts may also be flower extracts. Non-limiting examples of flower extract include hibiscus camomile, elder flower, lavender or linden flower.

  The plant extract may also be a fruit extract. Plant parts may, for example, be dried or raw herbs such as hop pellets, dried or fresh flowers or fruits.

  The plant concentrate may be a fruit concentrate, such as fruit juice, which has been concentrated by the removal of water.

Non-limiting examples of fruits useful for fruit aroma, fruit extract or fruit concentrate include orange, apple, banana, lemon, passion fruit, mango, pineapple, pear, kumquat or zabone .

The flavor compound may also be quinine, for example in embodiments where the beverage is a tonic-like beverage.

Beverage Properties The present invention relates to a method for preparing a beverage. The beverage may be any cereal extract based beverage, for example the beverage may be a malt based beverage, eg a fermented malt based beverage. In particular, the beverage may be beer. The beverage may also be a non-alcoholic malt based beverage, such as maltina. The beverage may also be another non-alcoholic beverage.

  In a preferred invention embodiment, the beverage is a beer. This may be any type of beer known to the person skilled in the art. In one embodiment, the beer is, for example, a lager beer. The beer may also be a low alcohol beer.

  Preferably, before storage, the beverage contains low levels of amino acids. Thus, the beverage may have at most 100 mg / L, more preferably at most 50 m / L, still more preferably at most 25 mg / L, even more preferably at most 10 mg / L, even more preferably at most 5 mg / L amino acids, methionine, phenylalanine It is preferred to have a total content of valine, leucine and isoleucine. In addition, the beverage preferably has a content of methionine of at most 15 mg / L, for example at most 10 mg / L, for example at most 5 mg / L, for example at most 3 mg / L.

  However, acceptable levels of Strecker amino acids can vary between beverages. Thus, in some embodiments of the invention, in particular in embodiments of the invention where the content of amino acids is reduced with the aid of REED treatment, it may be tolerated that the level of amino acids is somewhat higher than indicated above. . Thus, in some embodiments of the invention, the beverage has a total content of at most 200 mg / L, more preferably at most 150 m / L, even more preferably at most 100 mg / L of amino acids methionine, phenylalanine, valine, leucine and isoleucine. It can have.

It is also preferred that the beverage emits little ripening flavor. Thus, preferably, the beverage has a total maturation score of at most 2, for example at most 1.5 after storage for 2 weeks at 37 ° C. It is also preferred that the beverage has a total maturation score of at most 2, for example at most 1.5, after storage at 20 ° C. for 2 months. It is also preferred that the beverage has a total maturation score of at most 2, for example at most 1.5 after storage for 4 months at 20 ° C. It is also preferred that the beverage has a total maturation score of at most 2, for example at most 1.7 after storage for 6 months at 20 ° C. The total maturation score is determined on a scale of 0 to 5 according to a trained taste research group of at least 10 individuals, 0 being "non-aging" and 5 being "strongly aging". Preferably, said total maturation score is determined as described herein below in Example 4.

EXAMPLES The invention is further illustrated by the following examples, which however should not be construed as limiting the invention.

Example 1
Preparation of Glucose Wort 14.5% P wort with high glucose content was produced by mashing standard Pilsner malt with commercial brewing enzymes. The ground Pilsner malt was mashed with standard brewing water at 63 ° C. at a ratio of 1 kg malt to 3 liters of water. Immediately after mixing the ground malt with water, a commercial enzyme preparation containing alpha-glucosidase, alpha-amylase, and marginal dextrinase activity was added. They can convert carbohydrates and oligosaccharides to glucose. Calcium chloride was also added and the pH was adjusted to about 5.2 by the addition of phosphoric acid. After 30 minutes at 63 ° C., the temperature is increased at a rate of 1 ° C./min to 70 ° C., maintained at 70 ° C. for 60 minutes, increased at a rate of 1 ° C./min to 78 ° C. and finally 5 at 78 ° C. Maintained for a minute. The mash was then filtered and sparged to give a total volume of sweet wort about 75% higher than the original volume of water added to the malt. The sweet wort was adjusted to pH about 5.2 by the addition of phosphoric acid and calcium chloride was added. The wort was then boiled for 70 minutes. During this period some water was evaporated and the total volume of boiled wort was about 67% higher than the original volume of water added to the malt. Thus, a total of about 5.0 liters of boiled wort was obtained from 1 kg of malt. After the whirlpool process to remove sediment, the boiled wort was filled into kegs and maintained at 5 ° C. until further processing. This wort, and essentially the same produced wort, may be referred to herein as "glucose wort".

REED assisted enzyme conversion of glucose to gluconic acid Experimental lab-REED setup The experimental setup is a Biostat B fermentation system (B. Braun Biotech) for optional control of temperature, oxygen level, agitation rate and initial pH adjustment with KOH. International, currently Sartorius Stedim Systems, DE) required a 5 L bioreactor tank integrated. In a closed feed circuit, two REED membrane stacks (Jurag Separation, JS-9 from DK) were connected in parallel to the bioreactor. One stack, AX-REED, is an end membrane with 6 anion exchange membranes (Ionics, AR103-QDP from US) and 2 cation exchange membranes (DuPont, Nafion N117 from US) between feed and dialysate compartments There were three cell pairs to be used as Each cell pair had an active membrane area of 915 cm ² and contained a set of 2 mm thick serpentine path membrane spacers. One cell pair as CX-REED (JS-9) in the other stack, two cation exchange (CMB-sb from Astom, JP) and two end membranes (Fumatech, DE from FA) from both sides of the feed compartment Provided, one between the dialysate compartment and the electrode compartment at each end of the stack. Through two separate circuits in AX-REED and CX-REED, respectively, were fed dialysate 3-10L range of 0-1M KOH and 0-1M H ₃ PO _4. In both stacks, an electrode rinse solution consisting of 3 L of 1 M sodium hydrogen phosphate, neutral pH, was circulated using a centrifugal pump (Eheim, 1260 from Eheim, DE). Three separate pumps (FH100 from Fisher Scientific, US) were used for feeding and circulation of the dialysis solution. Current control and data logging for the two stacks was performed with a REED control module (Jurag Separation, model 2008.01-1500 from DK) connected to a laptop PC.

Using the equipment described above, it is possible to control the pH of the acid product mixture by exchanging the anion with OH ⁻ using AX-REED. Another possibility is to desalt the mixture by exchanging the cation with H ⁺ with CX-REED and the anion with OH ⁻ with AX-REED. Finally, CX-REED can be used to acidify the mixture simply by exchanging the cation for H ⁺ .

Experimental Procedure and Results 5 L glucose wort was transferred to a bioreactor and heated to 40 ° C. The wort had a conductivity of 2.27 mS / cm ² and the pH was 5.2. Oxygen was supplied from a pressure tank (52% oxygen), stirred and dispersed in the reactor by maintaining 30% oxygen saturation. At time = 0, 1 ml aliquots of the enzyme preparation containing both glucose oxidase activity and catalase activity were added. A gradual drop in pH was observed due to the conversion of glucose to gluconic acid and hydrogen peroxide. After about 20 minutes, when the pH reached 4.2, an automatic titration with 46% KOH was started to maintain the pH at 4.2. Time = 1.83 hours after enzyme addition, the conductivity increased to 4.82 mS / cm, the REED system was connected and took over pH control at pH 4.2. The AX dialysis solution was 8 L of 0.5 M KOH and the CX dialysate was 3 L DI water. At time = 2.25 hours, 6.33 hours, and 10.42 hours, a fresh 1 mL aliquot of enzyme was added. After 9 hours, the conductivity was about 6 mS / cm and desalting was initiated by adding 100 ml of H ₃ PO ₄ to the CX dialysate. At 11 hours, the test was completed with a pH of 4.2 and a conductivity of 4.17 mS / cm.

  Aliquots were taken for analysis throughout the study (A). The fresh sample contained a small amount of hydrogen peroxide but never exceeded about 50 ppm estimated using peroxide test strips from Merckoquant (Merck). After peroxide testing and conductivity measurements, the samples were heated to 80 ° C. for 10 minutes to inactivate the enzyme activity and then centrifuged at 4000 rpm for 10 minutes. Hydrogen peroxide could not be detected after this treatment. The sample was frozen until further analysis.

  The samples from Test A were analyzed for free amino acids by standard HPLC method. Analysis showed that the content of amino acids was significantly reduced during the test. Fejl! Henvissingskilde ikke fundet. 7 shows a reduction in the content of methionine, valine, isoleucine, leucine and phenylalanine in wort during test A. These amino acids are precursors to aging aldehydes that appear during storage in beverages.

  The experiment was repeated using essentially the same procedure. However, the pH was increased to 5.5 by titration with 46% KOH and the pH setpoint during REED processing was maintained at 5.5 throughout the test. This test (B) was also carried out at 40 ° C. and ended after about 11 hours. Sampling was performed as described in Example 1 and frozen for further analysis.

  Glucose wort and final products from Test A and Test B were analyzed for free amino acids by standard HPLC method. Analysis showed that the content of amino acids was significantly reduced during each of the two tests. FIG. 2 shows the recovery of amino acids in the two final products relative to glucose wort. Interestingly, the level of amino acids is even lower in samples treated with pH 4.2 compared to pH 5.5, a pH close to or higher than the pI of the amino acid in question is not required and probably not advantageous Is shown.

The contents of methionine, valine, isoleucine, leucine and phenylalanine in the final product are of particular interest for flavor stability, since these amino acids are precursors to aging aldehydes which appear during beverage storage It is from. The content of each of these amino acids in the wort and final product, and the data for their total are listed in Table 2.

  It is expected that the level of amino acids could be further reduced by carrying out REED treatment for a longer time.

  The final product was bottled and stored at room temperature for 13 months. The stored beverage during trial B was tasted by a research group of 5 trained beer tasters and the taste was judged as "not aged".

Example 2
The setup described in Example 1, the experimental lab-REED setup, was used for the fermentation of glucose wort by lactic acid bacteria. The bioreactor is filled with 5 liters of glucose wort prepared as described in Example 1, heated to 37 ° C., and 20 g of a commercial Lactococcus lactis culture (Chr. Hansen, DK, R-607). Strain). A gradual drop in pH was observed due to microbial conversion of glucose to lactic acid. Time = At 1.25 hours post inoculation automatic titration with 46% KOH to a setpoint of 5.5 was initiated. After 4 hours, the conductivity increases to about 5.0 mS / cm, start all AX-REED treatment pumps and add 200 ml of 46% KOH to 8 L DI water in AX-dialysate tank The pH control at pH 5.5 was then taken over by the REED control module. Three liters of DI water was used as CX-dialysate. The REED control module adjusted the current density through AX-REED in response to changes in pH. Thus, lactic acid OH ^- controlling the rate of exchange with, thereby, to control the pH. After 22.67 hours, when the conductivity is slightly higher than 10 mS / cm, 200 ml of H ₃ PO ₄ is added to the CX-dialysate and a fixed current is applied through the CX-stack to start the CX-REED process The desalting was initiated by After 24.75 hours, the acidification step was initiated by cutting the current to AX-REED and replacing AX-dialysate with DI water. The test was finished after about 25.5 hours, at which time the pH decreased to 4.35 and the conductivity was 1.7 mS / cm.

  Aliquots were taken for analysis throughout this test (C). All samples were centrifuged at 4000 rpm for 10 minutes and then pasteurized at 64 ° C. for 30 minutes. Samples were measured for Plato and the rest were finally frozen until further analysis.

  Samples were analyzed for free amino acids by standard HPLC method. The analysis showed that the content of amino acids was reduced to very low levels during the test. FIG. 3 shows Lc. Figure 7 shows the reduction of methionine, valine, isoleucine, leucine and phenylalanine content in wort during REED assisted fermentation of glucose to lactic acid using lactis. These amino acids are precursors of aging aldehydes that appear in beverages during storage. It is clear that the content of the 5 amino acids starts to decrease at the beginning of the REED-process, and that at the end of the test there is no or very little amount left.

  A pilot scale REED fermentation was also performed essentially as described for laboratory scale fermentation.

In one test (PT 58), a REED pilot system (Model 2011.01, equipped with 12 cell pair AX-REED stack and 10 cell pair CX-REED stack (the same membrane type as used in Example 1) The Jurag Separation, DK) was connected to a 50 L fermenter. The fermentor was filled with 50 L glucose wort, reduced to 30 ° C., and inoculated with 200 g of L. lactis at time = 0. The glucose wort had a pH of 5.2 at the start and a conductivity of about 2.5 mS / cm. Titration with 46% KOH to a pH setpoint at 5.5 was started at time = 0.83 hours. At time = 3.75 hours, the conductivity was about 5.0 mS / cm ² . The base titration was then discontinued and the REED system took over pH control. At time = 43.75 hours, the desalting step was started. At time = 44.25 hours, the acidification process was started. The REED fermentation was stopped at time = 45 hours.

  In another test (PT69), the REED pilot system is equipped with a 40 cell pair AX-REED stack and a 10 cell pair CX-REED stack (same membrane type as used in Example 1), in a 200 L fermentor It was connected. The fermentor was filled with 150 L of glucose wort, reduced to 30 ° C. and inoculated with 500 g of L. lactis at time = 0. The glucose wort had a pH of 5.2 and a conductivity of about 2.5 mS / cm at the beginning.

  The titration to pH setpoint at 5.5 with 46% KOH was then initiated. At time = 3.25 hours, the conductivity was about 5.0 mS / cm. The base titration was then discontinued and the REED system took over pH control. The desalting step was started at time = 27.15 hours. The acidification step was started at a time of 29.25 hours. The REED fermentation was stopped at time = 30.00 hours. Figure 4 shows the reduction of methionine, valine, isoleucine, leucine and phenylalanine content in wort during test PT69.

From test 69 described above, a reduction in the content of some other amino acids can be observed (possibly as a result of consumption by Lactococcus lactis), methionine, valine, isoleucine, leucine, It is clear that the content of and phenylalanine is maintained at pH 5.5 by titration with base and is hardly reduced in the initial phase, not by the REED system. However, the contents of methionine, valine, isoleucine, leucine and phenylalanine begin to decrease once the REED process is initiated. Depending on the processing time, these amino acids can be practically completely removed. See Table 3.

Example 3
The setup described in Example 1, an experimental lab-REED setup, was used to control pH during titration of glucose wort with lactic acid in this test (Test D). The bioreactor is filled with 5 L glucose wort prepared as described in Example 1, heated to 40 ° C., titrated with 80% food grade lactic acid (Galactid Excel 80 from Galactic) directly into the fermentor , Started at t = 0 hours. The pH was controlled at 5.5 by addition of 46% KOH via a Biostat control system. When the conductivity increased from about 2 mS / cm to about 5 mS / cm at t = 0.34 hours, control of pH was handed over to the REED control module to initialize AX-REED. Eight liters of 1.0 M KOH was used as AX-dialysate and 3 liters of DI water was used as CX-dialysate. At t = 4.34 hours, desalting is initiated by initiating CX-REED by adding 400 ml of 75% H ₃ PO ₄ to the CX dialysate, acidification at t = 6 hours , AX dialysate replaced with 3 L DI water, initiated by lactic acid titration, as well as interrupting the current on AX REED. Overall, 600 g of 80% lactic acid was titrated into the bioreactor. The test was discontinued at t = 6.75 hours when the pH reached 4.3. FIG. 5 shows the reduction of methionine, valine, isoleucine, leucine and phenylalanine content in wort during test D. Table 4 shows the absolute values for the untreated glucose wort and for the end of test D.

Example 4
Various doses of hydrogen peroxide (H ₂ O ₂ ) were added to an aliquot of 200 mL of glucose wort, prepared essentially as described in Example 1, pH 5.2. The samples were then incubated at 40 ° C. The concentration of H ₂ O ₂ at the beginning of the experiment was approximately 50 ppm, 250 ppm, 1000 ppm, and 2500 ppm, as estimated by the Merckoquant peroxide test strip (Merck). As a control, glucose wort without H ₂ O ₂ addition was also incubated. 5 mL samples were obtained after incubation between 2.25, 6.5 and 27 hours. The content of H ₂ O ₂ was estimated using peroxide test strips. The samples were then incubated with 10 μL of catalase suspension (C30-500 MG from Sigma) for 10 minutes at 40 ° C. to convert H ₂ O ₂ to water and oxygen. After this treatment, H ₂ O ₂ was no longer detectable and samples were stored frozen until analysis for free amino acids using standard HPLC procedures. Untreated wort was also analyzed for free amino acids and H ₂ O ₂ .

Recovery for valine, isoleucine, leucine, and phenylalanine is 80-105% in the presence of the highest tested concentration of H ₂ O ₂ , even after 27 hours, between recovery and H ₂ O ₂ concentration There was no correlation with Thus, H ₂ O ₂ has no effect on these four amino acids at the test concentration.

The methionine recovery after 2.25, 6.5 and 27 hours was 103%, 100% and 80% in the control sample, but 73% in the sample receiving 50 ppm H ₂ O ₂ at the start, The samples that received 42% and 10% and 250 ppm H ₂ O ₂ at the start were only 5%, 0% and 0%. Thus, even low doses of H ₂ O ₂ destroy methionine. Methionine could not be detected in all samples obtained during incubation with higher doses of H ₂ O ₂ .

Interestingly, no increase in methional content was detectable after incubation with H ₂ O ₂ . Thus, it is believed that methionine is not converted to streckeraldehyde methional.

FIG. 6 shows the content of amino acids, methionine, valine, isoleucine, leucine, and phenylalanine in those samples where H ₂ O ₂ is either at 0, 50, or 250 ppm initially.

The results for hydrogen peroxide are listed in Table 5. The consumption of H ₂ O ₂ during 27 hours at 40 ° C. was about 50 ppm.

Example 5
For the preparation of the beverage, two REED fermentations of Lactococcus lactis of glucose wort were carried out at pilot scale (50 L each) essentially as described in Example 2. One of the REED-fermentations was stopped after about 27 hours and the other was stopped after about 48 hours. After fermentation, the product was filtered to remove lactic acid bacteria. The filtered liquids were blended to obtain a sweet taste and flavored by dry hopping with a mixture of five different hop varieties. The total dose of hop pellet was 2 g / L and the pellet was left in the blend for 24 hours at 2 ° C. After dry hopping, the liquid was filtered and a small amount (about 5%) of aromatic beer (pale ale style) was added. The liquid was then carbonized, bottled and pasteurized. The final beverage was called REED-Beverage DS.

The content of free amino acids in the final beverage was determined by standard HPLC method. Data for methionine, valine, isoleucine, leucine and phenylalanine are listed in Table 6. These amino acids are precursors of senescent aldehydes. It is clear that the REED-beverage contains much lower amounts of these amino acids, in particular methionine, than the approximately 5.0% by volume alcohol (ABV) all-mol regular beer.

REED-Beverage DS was stored at 20 ° C. After storage for 2 and 6 months, the beverage was tasted by a trained beer taster. The research group is asked to assign the score to a paper-like, oxidation, ripening, bread-like, caramel, and burnt ripening flavor on a scale of 0-5, and finally the total ripening score Assigned. It is desirable to get a low score for all these flavors. In particular, it is desirable that these scores do not increase significantly during storage. The descriptors for the scoring system are listed in Table 7.

The total maturation score for REED-beverage DS was very low in both sessions, as listed in Table 8. For comparison, a typical score for all malt regular beer, about 5.0% ABV is included in the table. The scores for the individual ripening flavors are shown in FIG. The beverages scored very low for all ripening flavors and there was no significant difference between the scores obtained after 2 and 6 months. Thus, the individual ripening flavor was less pronounced for six months than for two months.

Example 6
In another test (PT 87), a 40 cell pair AX-REED stack (same film type as used in Example 1) and a 3 cell pair CX-REED stack (the same film type as used in Example 1) REED pilot system (Model 2011.01, Jurag Separation, DK)) was connected to a 50 L fermenter. The fermentor was filled with 10 kg maltose wort, 17.6 kg glucose wort, and 22.4 kg water to obtain a final plate of 8%. Maltose wort was the conventional wort. Glucose wort was produced essentially as described in Example 1. The fermentor was heated to 37 ° C. and inoculated with 200 g of Lactococcus lactis at time = 0. The glucose wort had a pH of 5.2 and a conductivity of about 1.5 mS / cm at the start. Titration with 11.5% KOH to a pH setpoint of 6.0 was started at time = 0 hours. At time = 1.5 hours, the conductivity was about 5.0 mS / cm. The base titration was then discontinued and the AX-REED system took over pH control. At time = 11 hours, the desalting step was started (using AX-REED and CX-REED simultaneously). At time = 13 hours, the acidification step was started (CX-REED only). The REED fermentation was stopped at time = 13.5 hours.

  FIG. 9 shows the reduction of amino acid concentration during fermentation. The Strecker aldehyde forming amino acids are all removed after 13 hours of fermentation.

  The fermentation broth was filtered to remove cells, carbonized, bottled and stored at three different conditions: 2 ° C, 20 ° C or 37 ° C. The beverages were tasted and scored by trained study staff as described in Example 5. Beverages score very low for all ripening flavors, 2 weeks at 2 ° C (score: 1.4), 2 weeks at 37 ° C (score: 1.6), or 3 months at 20 ° C (score) : 1.6) There was no significant difference between the total maturation scores obtained after. The descriptors for the scoring system are listed in Table 7.

Example 7
In yet another test (PT97), the REED pilot system described in Example 6 was connected to a 50 L fermentor. The fermentor was filled with 50 kg of plato 14.3% glucose wort, prepared essentially as described in Example 1. The fermentor was heated to 30 ° C. and inoculated with 20 g of L. lactis at time = 0. The glucose wort had a pH of 5.2 and a conductivity of about 2.5 mS / cm at the beginning. The titration to a pH setpoint of 5.5 with 11.5% KOH was initiated at time = 0 hours. At time = 4.2 hours, the conductivity was about 5.5 mS / cm. The base titration was then discontinued and the AX-REED system took over pH control. At time = 24 hours, two 5 L samples (Sample A and Sample B) were taken from the fermenter and the desalting step was then started by initiating CX-REED. At time = 26.5 hours, the acidification step was initiated by cutting off AX-REED. The REED fermentation was stopped at time = 28 hours. At this time, the pH decreased to 4.35 and a third 5 L sample (Sample C) was taken from the fermentor.

  Immediately after sampling, sample A was transferred to a 30 ° C. chamber and gently stirred. A gradual drop in pH was observed due to the formation of lactic acid by lactic acid bacteria in the sample. When the pH decreased to 4.35, sample A was centrifuged and sterile filtered to remove lactic acid bacteria. The samples were finally bottled and pasteurized at 64 ° C. for 30 minutes.

  Immediately after sampling, sample B was centrifuged and sterile filtered to remove lactic acid bacteria. The samples were then transferred to a 5 L bioreactor tank integrated in the Biostat B fermentation system described in Example 1. Oxygen was supplied to the bioreactor from a pressurized tank (52% oxygen) and dispersed in the liquid with agitation to maintain 30% oxygen saturation. A 0.5 mL aliquot of enzyme preparation containing both glucose oxidase activity and catalase activity was added to the liquid. After addition of the enzyme preparation, a gradual drop in pH was observed due to the conversion of glucose to gluconic acid and hydrogen peroxide. The oxygen supply was terminated when the pH reached 4.35. Sample B was then heated to 80 ° C. and maintained at this temperature for 30 minutes to inactivate the enzyme activity. The samples were finally bottled and pasteurized.

Immediately after sampling, sample C was centrifuged and sterile filtered to remove lactic acid bacteria. A 1 L aliquot was then removed and 1 ml of 30% H ₂ O ₂ was added to this sample (Sample D). Sample D was left at about 22 ° C. for 3 days, then bottled and pasteurized. The remainder of Sample C, except bottling and pasteurization, did not receive further processing.

The content of free amino acids in the four samples was determined by standard HPLC method. Table 9 shows the contents of methionine, valine, isoleucine, leucine and phenylalanine. These amino acids are precursors to aging aldehydes which appear by strecker decomposition during storage.

  It is expected that the level of amino acids could be further reduced by carrying out REED treatment for a longer time.

  One bottle of each sample is stored at 37 ° C. for 2 weeks, while the remaining bottle is stored at 5 ° C. The aroma and taste of the warm and stored samples are then evaluated by a trained beer taster study group to determine the level of strecker aldehyde.

Claims (22)

A method for producing a flavor stable cereal based beverage, said method comprising
i) providing a cereal extract comprising at least 25 mg / L methionine;
ii) The cereal extract is treated to reduce the content of one or more amino acids selected from the group consisting of methionine, phenylalanine, valine, leucine and isoleucine according to a method comprising the following steps: Obtaining a cereal extract;
a) increasing the level of acid and / or base in the cereal extract, and b) reversing at least a portion of the acid anion of the increased acid and / or the basic cation of the increased base. Removing through an electrically enhanced dialysis (REED) membrane stack, or treating the cereal extract to reduce methionine content by incubating the cereal extract with H ₂ O ₂ ,
iii) processing the processed cereal extract into a beverage;
The method, wherein the beverage has a total content of at most 100 mg / L methionine, phenylalanine, valine, leucine and isoleucine and / or a content of methionine of less than 5 mg / L. A method for reducing the content of at least one amino acid in cereal extract, comprising
a) increasing the level of acid and / or base in the cereal extract; and b) reversing at least a portion of the acid anion of the increased acid and / or the basic cation of the increased base. Removing through an Electrically Enhanced Dialysis (REED) membrane stack.   3. The method of claim 2, wherein the level of acid is increased using an enzyme or mixture of enzymes capable of catalyzing the formation of acid.   The method according to claim 3, wherein the enzyme is glucose oxidase.   3. The method of claim 2, wherein the level of the acid is increased using a microorganism capable of fermenting sugar to form an organic acid.   The method according to claim 5, wherein the microorganism is Gluconobacter. I. Inserting the cereal extract into a tank connected to the AX-REED membrane stack and the CX-REED membrane stack;
II. Increasing the level of acid in the cereal extract;
III. Removing at least a portion of the acidic anions of the increased acid in the cereal extract by AX-REED treatment;
IV. Removing at least a portion of the cations in the cereal extract by CX-REED treatment,
The method according to claim 2, wherein the steps II, III and IV are performed sequentially, partially simultaneously or simultaneously. The REED process is in said processed cereal extracts, at most 100 mg / L, at most 50 m / L, at most 25 mg / L, at most 10 mg / L, methionine at most 5 mg / L, phenylalanine, valine, leucine and isoleucine total The method according to claim 2, which is carried out for a sufficient time to obtain the content. i) providing a cereal extract comprising at least 25 mg / L methionine;
ii) inserting the cereal extract into a tank connected to the AX-REED membrane stack and the CX-REED membrane stack;
iii) increasing the level of acid in the cereal extract using a microorganism capable of fermenting the sugar to form an organic acid;
iv) removing at least a portion of the acid anions of the increased acid in the cereal extract by AX-REED treatment;
v) acidifying the liquid;
vi) thereby obtaining a processed cereal extract;
The method according to claim 1, further comprising the step of: vii) further processing the processed cereal extract into a beverage.   10. The method according to claim 9, wherein the acidification is carried out by treatment with an enzyme or mixture of enzymes capable of catalyzing the formation of an acid.   The method according to claim 9 or 10, further comprising the step of treating the cereal extract with an oxidizing agent, said step being carried out after step vi). A method for reducing the content of methionine in cereal extracts, comprising
i) A method comprising the step of incubating the cereal extract with H ₂ O ₂ . Step ii) comprises or consists of the following method:
The method according to claim 1, comprising i) incubating the cereal extract with H ₂ O ₂ . A method for producing a flavor stable cereal based beverage, said method comprising
i) providing cereal extract;
ii) treating the cereal extract to reduce the content of at least one amino acid according to the method comprising the following steps, thereby obtaining a treated cereal extract;
a) increasing the level of acid and / or base in the cereal extract, and b) reversing at least a portion of the acid anion of the increased acid and / or the basic cation of the increased base. Removing through an electrically reinforced dialysis membrane stack,
Or c) obtaining a treated cereal extract by incubating the cereal extract with H ₂ O ₂ ,
iii) processing the processed cereal extract into a beverage;
Method, including.   The method according to any one of claims 1 to 11, wherein the cereal extract is wort, malt based wort or glucose wort.   The method according to claim 12, wherein the cereal extract is wort, malt-based wort or glucose wort.   The method according to any one of claims 1, 9, 11, 14 and 15, wherein step iii) comprises microbial fermentation of the treated cereal extract.   A method according to claim 9 or 11, wherein step vi) comprises microbial fermentation of the treated cereal extract. Step iii) a) adding additional compound to the treated cereal extract, and / or b) mixing the treated cereal extract with another liquid c) the treated cereal extract and the microorganism The method according to any one of claims 1, 9-11 and 14-18, comprising the step of fermentation according to Step vi) a) adding additional compound to the treated cereal extract, and / or b) mixing the treated cereal extract with another liquid c) the treated cereal extract and the microorganism The method according to claim 9 or 11, comprising the step of fermentation according to   The method according to any one of claims 1, 9-11 and 14-20, further comprising the step of storing the beverage for at least one week. The method according to claim 9 or 11, further comprising storing the beverage for at least one week.

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